Molecular Bioengineering - Industrial & Engineering Chemistry

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Molecular Bioengineering Arul Jayaraman, Martin L. Yarmush,* and Charles M. Roth† Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, Harvard Medical School, and Shriners Burns Hospital, Boston, Massachusetts 02114

Molecular bioengineering is an emerging discipline that draws from advances in science and engineering to seek molecular-level solutions to complex problems in medicine and biotechnology. Based on tremendous advances in our understanding of biology and physiology, a number of promising molecular diagnostics, therapeutics, and biotechnological solutions are under development, but the properties of the molecules used in such techniques often require some modifications to satisfy the constraints of a particular application. In this review, we present an integrative view of how one might address problems in molecular bioengineering. We present the relevant properties of base constituent molecules (proteins, nucleic acids, small organics), describe some of the design principles that are determinants of effectiveness for various applications, review some of the tools of modern molecular biology that are being used to address these problems, and discuss some of the means being used to assess potential solutions accurately and rapidly. Taken together, these tools and approaches are likely to play a significant role in improving the development of biological molecules for applications in biotechnology and medicine. Introduction Molecular bioengineering encompasses a broad swath of approaches used to solve biological problems by developing a fundamental understanding of interactions among various molecules and using this information to design novel molecules or to improve upon existing ones. By virtue of its broad scope, molecular bioengineering is interdisciplinary and involves principles of engineering, biology, chemistry, and physics in its applications. Biomolecules such as enzymes have been manipulated for improved performance for many years. However, optimization has been hindered by the complex relationship among molecular properties (e.g., amino acid sequence and three-dimensional structure), design principles (e.g., turnover rate vs specificity), and functional activity (e.g., yield of an enzyme-catalyzed reaction). With developments in technology and informatics, a rational basis for engineering biologically important molecules is now becoming feasible, one that envisions a combination of theoretical (e.g., rational molecular design, structural modeling, computational genomics) and experimental (e.g., site-directed mutagenesis, combinatorial chemistry, molecular evolution) approaches in alter properties of the molecule that are of interest to specific application(s). At the molecular level, the design principles for biological processes involve fundamental principles of physical chemistry and engineering sciencesparticularly thermodynamics and kineticss applied to biochemical reaction cycles and networks. Furthermore, engineers are well-suited to analyzing the tradeoffs involved when an improvement in one property of a biological molecule has an adverse effect on other properties. As a result, there is a great opportunity * To whom correspondence should be addressed to at Shriners Hospital for Children, 51 Blossom Street, Boston, MA 02114. Tel.: 617-371-4882. Fax: 617-371-4950. E-mail: ireis@ sbi.org. † Departments of Chemical and Biochemical Engineering and Biomedical Engineering, Rutgers University, 98 Brett Road, Piscataway, NJ 08854-8058.

for chemical (or biochemical or bio-) engineers, armed with sufficient knowledge of the tools of molecular biology, to contribute to the development of molecular bioengineering. The need for molecular bioengineering approaches is inherent in the increasing need for improved molecules in a variety of industrial, research, and medical applications, which, in turn, are driven by economic as well as social considerations. Advances in a variety of applied chemical and biological technologies are resulting in increases in the number of viable drug targets and drug candidates. Often, however, drug candidates that are favorable based on some criteria are not acceptable on others. For example, mouse monoclonal antibodies often have favorable pharmacokinetic profiles and excellent pharmacological properties (i.e., fairly high-affinity binding with excellent specificity), but their immunogenicity is unacceptable for therapeutic purposes. In this case, strategic alterations might result in a modified antibody whose adverse effects are eliminated without its biological effectiveness being compromised. The specific technique used for modification will depend on both the nature of the desired modification and the biochemical identity of the original molecule (i.e., gene or protein or other). A critical determinant of the success of any strategy is the ability to assay for the desired functionality rapidly but in a way that is meaningful. For example, the immunogenicity of humanized monoclonal antibodies is difficult to assess via in vitro assays or even in vivo experiments in animals. This review encompasses a host of topics that individually have been the focus of detailed reviews, which provide valuable detailed information on techniques and applications. Our goal here is to provide an overview of these techniques, with particular emphasis on developing a strategy that relates the desired functional improvement to specific design criteria, modification techniques, and evaluation assays. There are many applications to which molecular bioengineering principles could be applied. Of these, most of our discussion focuses on a set of representative applications, which

10.1021/ie0102549 CCC: $22.00 © 2002 American Chemical Society Published on Web 08/30/2001

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Ind. Eng. Chem. Res., Vol. 41, No. 3, 2002

Table 1. Use of Molecular Bioengineering Techniques in Selected Applications application

design criteria

base molecules

techniques

methods of evaluation

enzyme-catalyzed organic reactions

reaction kinetics protein stability product specificity

proteins

mutagenesis directed evolution combinatorial chemistry

functional assays phage display high-throughput screening

improvement of protein folding yield

kinetics of folding protein stability

proteins

mutagenesis

functional assays binding assays

generation of novel antimicrobials

affinity for target physiological distribution

peptides small organics

directed evolution phage display combinatorial biosynthesis combinatorial chemistry conjugation (chimeras)

phage display high-throughput screening pharmacological assays

monoclonal antibody therapeutics

affinity for target kinetics of target recognition specificity for target physiological distribution

proteins

mutagenesis conjugation (genetic and chemical)

functional assays binding assays

antisense oligonucleotide therapeutics

affinity for target kinetics of target recognition stability (nuclease resistance) specificity for target physiological distribution

nucleic acids

combinatorial synthesis chemical conjugation chemical modification

binding assays functional assays microarrays

gene therapy

stability physiological distribution

nucleic acids

directed evolution genetic engineering molecular conjugation

functional assays pharmacological assays

are listed in Table 1. For some of these examples, such as the development of antisense therapeutics (Figure 1), molecular bioengineering can potentially play a role at multiple points along the development pipeline. Application of the engineering paradigm early in development might aid in reducing the transitions from drug discovery to development and to manufacturing stages, thus reducing the costs and duration of overall drug development efforts. These examples provide a range both in terms of constituents and approaches and are intended to provide a basis for integration across the facets of molecular bioengineering, rather than serving as an exhaustive compendium. A. Base Molecules The choice of base molecule for molecular bioengineering can be dictated by the problem of interest (e.g., nucleic acid for gene therapy) or be flexible (e.g., proteins, peptides, nucleic acids, and small organics might each be selected as ligands for binding arbitrary drug targets). In either case, the biological and physicochemical properties of the constituents dictate how they can be manipulated. In this section, we briefly discuss the parameters that characterize these biomolecules, approaches to redesigning them, and their consequences. A.1. Nucleic Acids. Nucleic acids are extremely attractive candidates for modification because they represent the genetic code and slightly altering the properties of a single gene markedly changes the expression or properties of the specific gene product. Depending on the structure (robustness) of the biochemical network in which the gene and its encoded protein are acting, genetic manipulations can have a profound or very minor impact on phenotype. Nucleic acids (except RNA) are extremely stable molecules under ambient conditions, and this resilience makes

redesigning nucleic acids generally easier than modifying proteins. Furthermore, nucleic acids (with the exception of catalytic RNA) do not exhibit any enzymatic activity and do not require any three-dimensional structural information for their properties to be modified. Rather, the one-dimensional nature of the genetic code makes rational modification quite tractable, although, of course, there are technical challenges. The most commonly manipulated forms of nucleic acids are oligonucleotides (short 15-25 base fragments of singlestranded nucleic acids) and whole genes. Because oligonucleotides are synthesized chemically on solid supports, a variety of modifications can be incorporated. Virtually all oligonucleotides used in vivo (including cell culture) are modified to increase their resistance to nuclease attack, most commonly by altering their backbone chemistry or modifying their sugar moieties.1,2 A comprehensive list of oligonucleotide analogues that have been used for modification during synthesis has been compiled by Walton et al.3 Oligonucleotides have also been synthesized with linker molecules during synthesis for specific applications. For example, a steroid cyclic disulfide anchor has been used to conjugate oligonucleotides to gold particles for use as sensors.4 In addition to chemical modifications, the physical properties of oligonucleotides can be exploited or manipulated to impart desired properties. In particular, the uniformly distributed negative charge of oligonucleotides provides an opportunity for them to be efficiently packaged with positively charged cationic polymers or liposomes to generate neutral complexes. Because the cellular uptake of neutral complexes is generally more efficient than that of negatively charged oligonucleotides, this approach has been used effectively in both therapeutic and research applications. Apart from their physicochemical properties, the length and base composition of the sequence involved in hybridization have

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Figure 1. Molecular bioengineering in therapeutic molecule development. The integration of molecular bioengineering can be beneficial to the development of therapeutic molecules such as antisense oligonucleotides. From the identification of potential target sequences to improved synthesis methods, screening assays, packaging, and in vivo delivery, design principles such as affinity, kinetics, stability, and function can result in the improvement of the individual steps in the development process as well as in the overall efficacy of the therapeutic molecule.

also been manipulated to improve the affinity and specificity of the oligonucleotide for its target. In antisense applications, a broad optimum of 15-25 bases seems to exist, as that range has sufficient length for affinity and specificity, while not being so long that subsequences of the oligonucleotide would bind to nontargeted mRNAs. Manipulating a gene (encoding the protein of interest) to improve specific protein function has become a relatively simple procedure with the advent of recombinant DNA technology, and a detailed discussion of gene manipulation methods is beyond the scope of this review. The developments of inducible promoters for recombinant protein production, altered regulatory systems using anti-metabolites, and vaccine vectors that can carry large loads of DNA for gene therapy and the synthesis of rationally designed antibodies for use in bioseparations and therapeutics have all significantly impacted the way in which biomolecules have been produced and purified. Genes exhibit the same basic physicochemical properties as oligonucleotides, most notably one negative charge per backbone linkage. As a result, they can be packaged with the same types of cationic species, and with the growing interest in nonviral gene therapy, the resulting microstructures have begun to be investigated in some detail.5,6 A.2. Proteins. In general, protein engineering and genetic engineering can be considered as two sides of the same coin, as the final objective in both cases is to generate a protein with improved properties. However,

the methodologies for engineering proteins are not as simple or well-characterized as those for manipulating DNA. This is primarily due to the complex threedimensional structure of proteins; as a result, the effect of modifications on structure or function often cannot be predicted a priori. Furthermore, many proteins are more sensitive to handling than nucleic acids, and extensive manipulation or harsh environmental conditions can adversely affect their function after manipulation. Because proteins are encoded by genes, an easy way to manipulate proteins would be to utilize advances in genetic engineering to obtain protein variants.7,8 Particularly when performed in a high-throughput manner, this approach eliminates the need for structural information for functional improvement of proteins. Although this approach works well for single-subunit proteins (proteins that are synthesized from a single gene), it is not easily applied to multi-subunit proteins, where the fact that the individual subunits are encoded by different genes makes the required genetic engineering a nontrivial task. Several other properties of proteins make them amenable to manipulation. The ability of proteins to elicit immunogenic responses when introduced into host animals has been used to generate natural and recombinant antibodies against specific proteins of interest.9 Proteins can also be modified after their synthesis, and this property has been utilized to conjugate different moieties to their carboxyl, amino, and sulfhydryl groups.10,11 Site-specific modification or conjugation,

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although possible in certain circumstances, is generally quite difficult.11 A.3. Miscellaneous Organics. Advances in methods to manipulate molecules have also impacted the production of several organics including acids,12 esters and alcohols,13 polyketides,14 and antibiotics. Here, we mention two classes of molecules, namely, polyketides and antibiotics, that are particularly attractive for bioengineering. Polyketides are natural products produced by Streptomyces that have been used in the synthesis of antibiotics, anticancer agents, cardiovascular agents, and veterinary products.15 The synthesis of polyketides is directed by the activity of several enzymes in a modular or iterative manner, which allows the use of combinatorial synthesis methods to generate a wide variety of polyketides that have improved activity and a broader antimicrobial range.14 The current spectrum of available antibiotics (considered second- and thirdgeneration) is mainly produced by chemical modifications of approximately 15 base compounds and is targeted to 15 conventional bacterial targets.16 These chemical modifications allow the use of combinatorial methods to generate libraries of compounds and generate diversity in a manner similar to polyketide biosynthesis. Furthermore, the sequencing of various bacterial genomes is providing newer avenues for bioengineering in antibiotic synthesis. Sequence analysis and computational methods are being used to identify improved targets for existing antibiotics as well as to develop novel antibiotic molecules. B. Design Principles for Molecular Bioengineering With developments in technology and informatics, a rational basis for the engineering of biologically important molecules is becoming feasible. Utilizing these techniques to their maximum effectiveness requires a distillation of a complex outcome (e.g., therapeutic effectiveness) to one or several molecular criteria (e.g., binding affinity, physiological half-life, etc.). In this section, we present some of the most common design criteria on which biological molecules are modified and optimized. B.1. Affinity. Molecular recognition provides the means for biological information transfer among various cells, tissues, and organ systems. Binding affinity is the most direct, quantitative parameter describing the strength of interaction between two molecules, as it relates the equilibrium concentrations of bound and unbound species. Frequently, an affinity-controlled molecular recognition event is the first step in initiating a complex biological response. For example,17 the binding of cellular receptors by biological ligands followed by oligomerization is known to activate almost all membrane-bound receptors.18 These binding and multimerization processes result in the initiation of signaling cascades that eventually lead to a physiological response. Therefore, binding-affinity-based interactions at the cell surface are one crucial determinant of cellular activity, and control of binding affinities between ligands and receptors is one means by which to “engineer” a biological response.19 This concept has been applied to engineer weak affinity receptor binding events to result in an increased cellular immune response via increased binding interactions.20 Similarly, enhancing the binding affinity between antibodies and antigens has also

significantly improved the in vivo performance of antibodies as therapeutics.21 However, there are also numerous examples in which the biological response is not directly related to the affinity.18,22 For example, a number of variants of human growth hormone with improved affinity for its receptor failed to produce a significant change in signal transduction or cellular proliferation.18,19 The binding affinity between two biological macromolecules, particularly proteins, is determined by a number of physicochemical factors, including the hydrophobicity of the binding interfaces, electrostatic interactions, and shape complementarity between the binding regions. Except for a limited number of small ligands such as calcium or carbon monoxide, naturally occurring ligands are generally organic species for which affinity can be related to size. The free energy of binding has been correlated with the number of non-hydrogen atoms with a slope of -1.5 kcal/mol for relatively small ligands up to about 10 nonhydrogen atoms.23 After this, the affinity increases much more slowly, with a maximum in most cases of about -15 kcal/mol, corresponding to a dissociation constant of 10-12 M.23 The correlation with size corresponds to increasing area of contact between ligand and receptor, which, in the receptor, is usually organized in the form of a pocket or cavity.24 Size and shape contribute to binding affinity in two ways: through the burial of nonpolar surface area (the so-called hydrophobic effect) and through the accumulated strength of short-range van der Waals forces. Some differences in the relative magnitudes of these effects occur depending on whether the individual species are stable in the monomeric state.25 Electrostatic interactions are usually not major contributors to macromolecular binding because of the penalty of desolvating charged groups; however, in some instances, the charged groups in the ligand are distributed in an “optimized” manner, allowing for a favorable contribution to affinity.26 Nucleic acid hybridization is somewhat different, in that it is more specific and driven by hydrogen-bond formation between Watson-Crick base pairs and by stacking of adjacent nucleosides. B.2. Kinetics. Whereas affinity (more precisely, a chemical potential difference) describes the thermodynamic driving force for a molecular interaction, kinetics are critical to determining the extent to which the interaction is observed in practice. The kinetics of three types of molecular processes are of interest here: the kinetics of self-assembly (protein or RNA folding), the kinetics of association of two biological macromolecules, and the kinetics of enzyme-catalyzed reactions. The dynamics of behaviors involving integrated systems of biological molecules have already been mentioned and are considered separately (section B.5). The kinetics of formation of a properly folded protein are a critical determinant of the yield in recombinant bioprocesses and protein engineering. The competition between proper folding and the tendency to form folding intermediates or improperly folded protein aggregates is a delicate one governed by many factors, including the size of the protein to be folded, the existence and stability of folding intermediates, and the number of disulfide bonds to be correctly formed. Because the folded and unfolded states of the enzyme are unlikely to be at equilibrium on short time scales,27 engineering the kinetics between the folded and unfolded states of a protein can increase the fraction of correctly folded protein and even enhance enzymatic activity.


Biomolecular association reactions occur following collisions of correctly oriented constituents. The rate of association is primarily dictated by the diffusion-limited rate, between 109 and 1010 M-1 s-1, and the probability of the two molecules having the correct orientation to bind, which, for protein-protein association, typically lowers the association rate to about 105 M-1 s-1.28 For example, at a typical cellular concentration of 100 nM (roughly 105 molecules/cell), this rate corresponds to a characteristic time for association on the order of 100 s, which is the time scale over which many binding and catalysis events occur in cells, for instance, in signal transduction. This rate can be significantly enhanced by long-range electrostatic interactions. For example, the binding affinity of the bacterial ribonuclease barnase by its inhibitor barstar is increased by over 4 orders of magnitude by electrostatic attraction.29 Systematic mutagenesis studies of antibody-antigen interactions show that, in the absence of electrostatic enhancement, differences in affinity among homologous species are usually governed by differences in dissociation rate rather than in association rate.30,31 As a result, it is difficult to modify the association rate of biomolecular processes. Nucleic acid association rates are similar in order of magnitude (104-105 M-1 s-1), and for short stretches of nucleic acids, affinities are controlled by dissociation rates. However, for complex molecular interactions, such as antisense oligonucleotide binding coupled to partial unfolding of the target mRNA, affinities are controlled by the association rate,32 presumably because of the strong dependence on unfolding the RNA to provide an accessible site. Therefore, kinetic selection of antisense oligonucleotides might prove both feasible and useful. The classic example of interplay between affinity and kinetics is in the Michaelis-Menten expression describing the rate, v, for an enzyme-catalyzed reaction as a function of enzyme concentration, [E], and the substrate concentration, [S]

v)

kcat[E][S] Km + [S]

(1)

where kcat is the rate constant of the catalytic step (also called the turnover number, as it sets the maximum rate of the reaction if enzyme-substrate binding is fast relative to catalysis), and Km is the Michaelis constant, which is identified as the strength of enzyme-substrate binding but which, in fact, also involves an interplay of kinetic rates

Km )

kcat + koff kon

(2)

The rate constants kon and koff are the binding and dissociation rates, respectively, for enzyme-substrate binding, so that Km is equal to the equilibrium dissociation constant when catalysis is slow compared to dissociation but involves the competing effects of dissociation and catalysis in the general case. B.3. Stability. Whereas affinity and kinetics mainly impact the generation of biomolecules, the stability of the generated product is crucial for its functional activity as well as for long-term storage. The stability of a biological molecule encompasses its resistance to a range of processes, beginning with chemical reactivity with the local environment (e.g., oxidation, deamination)

and including biochemical degradation, physical stress, and physiological clearance for the case of in vivo therapeutics. Biomolecules produced for various applications are subject to an array of environmental conditions during production, processing, and storage, as well as in the final usage environment; altering molecular properties to enhance stability without compromising function is a major challenge. Although a number of techniques exist to alter the stability of a biomolecule, their effectiveness is often application-dependent, because of the thermodynamic consequences of enthalpy-entropy compensation.33 That is, many modifications can be made that provide energetic stabilization of a biomolecular structure or interaction, either through covalent linkage or through enhancement of electrostatic interactions, hydrogen bonding, etc. However, a consequence of stabilization is usually an increase in conformational order, resulting in a decrease in entropy. The net change in the free energy of protein denaturation, ∆(∆GD), upon modification can be expressed as

∆(∆GD) ) ∆(∆HD) - T∆(∆SD)

(3)

where ∆(∆HD) and ∆(∆SD) are the changes in the enthalpy and entropy of denaturation, respectively. For example, Robinson and Sauer introduced a disulfide bond into a single-chain version of the Arc repressor to increase its stability.34 Whereas the enthalpy of chemical denaturation (in guanidium hydrochloride) was increased by 18 kcal/mol, the free energy of denaturation decreased by 3.6 kcal/mol because of the entropic cost associated with the loss of conformational flexibility. As a result, the stabilized molecule is actually more susceptible to chemical denaturation. On the other hand, the thermal stability, which is characterized in terms of the melting temperature, Tm, of the disulfideengineered protein, was found to increase by 15 °C.34 This can be interpreted as reflecting a change in the heat capacity difference on unfolding due to the modification. The feasibility of successfully altering biomolecule stability also depends to a great degree on the environment in which the molecule is present and the extent of structural information available. In industrial applications, the environment in which an enzyme functions is radically different from that inside the cell; hence, adapting the enzyme to retain its active conformation in a harsh chemical and thermal environment can increase its stability and optimize its performance. For example, a chemical cross-linking between the 35th (glutamate) and 108th (tryptophan) residues has been shown to increase the unfolding temperature of lysozyme by 20 °C.35 Shaw and Bott27 have listed several other examples of amino acid substitutions that result in increased enzyme stability. The stability of oligonucleotides in antisense-mediated modulation of gene expression in vitro and in vivo is usually altered by modifying the backbone of oligonucleotides or nucleosides.36 Similarly, peptides and proteins have been altered by introducing disulfide bonds or generating cyclic molecules, each of which results in an increase in thermostability.37,38 A third issue in biomolecule stability arises from instability during handling and storage. This is especially true for pharmaceuticals, which might need to be stored for months and then readily reconstituted in an active form.

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B.4. Specificity. Specificity is an essential feature of biological processes, ranging from the isomeric synthesis of sugars and proteins to antigen recognition by antibodies to the fidelity of gene transcription. Enzymes exhibit a very high degree of specificity for the substrates that they catalyze, which can be altered for a variety of research and industrial applications. Altering the specificity of the enzyme (without any change in activity) can lead to an expansion or reduction in the number of substrates it recognizes.39 Decreasing the specificity can also be advantageous, for example, in promoting diversity in the products, as demonstrated by Ibba and Henecke in the synthesis of proteins with unnatural amino acids.40 Apart from altering the choice of specific substrates, coenzyme41 as well as product42 specificity are possible targets for bioengineering applications. Catalytic antibodies are another example of introducing a new function to antibodies to result in specific catalytic activity (similar to enzymes). For example, aldolase antibodies have been engineered to recognize a wide range of substrate structures.43 The specificity of biomolecules can also be of utility in recombinant bioprocesses where the efficient purification of the produced molecule is desired. In such cases, the specificity of antibodies for their ligands can be engineered for improved downstream purification strategies. B.5. Function in a Physiological Context. Whereas molecular bioengineering approaches target specific molecules and modify their properties, the target molecule is typically in a larger cellular or assay-specific context. For example, many cellular responses are elicited by ligand binding to a receptor on the cell surface, at which point signal transduction occurs through catalytic events triggered by binding, with ligand-receptor dissociation and receptor internalization serving as two mechanisms of modulating a response. As a result, a number of authors have described differences in dose-response curves for ligand binding and cellular effect in terms of kinetic processes.18,19,44 Most biomolecular events are organized into networks of binding and catalytic processes, whose overall properties depend on the integrated activity of the thermodynamics and kinetics of individual reactions. As a simple example, many transcription factors are synthesized and exist in cells as monomers (M), which form dimers (D) under activating conditions. Only the dimer binds to its cognate gene sequence (S) to form a complex (C), which promotes or represses gene expression. Under equilibrium conditions, this process can be described by the simple sequence KD1

M + M y\z D KD2

D + S y\z C

(4)

The concentration of dimer required to result in occupation of one-half of the DNA sequence sites is equal to the dissociation constant of the second step, KD2. The concentration of dimer can be related to the corresponding concentration of monomer via the first step, which has the dissociation constant KD1. As a result, the concentration of monomer that will result in occupation of one-half of the DNA sequence sites, which will be its

apparent dissociation constant, KD,app, is

KD,app ) xKD1KD2

(5)

Often, the first step has a weaker affinity (higher KD) than the second; therefore, the apparent affinity is weaker than that for transcription factor-DNA binding. As a result, generation of a fusion construct that eliminates the need for the first step can result in dramatic changes in the apparent dissociation constant45 (see section D.1.5). In many biomedical applications, the effectiveness of an engineered biomolecule depends on its interaction at multiple physiological sites. For example, the efficiency of a gene therapy protocol is closely linked to the cellular uptake and intracellular trafficking of the (viral or nonviral) vector, as well as to the expression by and/or integration of the gene into the host genome.46 In oligonucleotide therapeutics, the uptake of oligonucleotides, the subcellular trafficking, and the affinity of hybridization to their target in vivo each contributes to their low efficacy. Therefore, efficiency of the biomolecule in its cellular environment is an integrated design variable that involves contributions from several individual design variables. However, in the absence of complete structural information on biomolecules or mechanistic information (e.g., biochemical reaction network structure) on their cellular role, it can be difficult to identify specific design principles that are crucial to efficiency. B.6. Multifunctionality. Often in biological processes, a single design parameter (such as binding affinity or kinetics) cannot completely characterize a molecule’s properties. In these cases, multiple parameters must be modified and optimized to enhance the function of a biomolecule, a task that is extremely tedious and difficult. The notion that biomolecule function can be adequately described by small domains in the molecule has led to the concept of having several such domains in a single molecule, with each domain providing a particular function. The concept of multifunctionality has made it feasible to develop an optimal strategy for enhancing biomolecule function without optimizing several parameters individually. One application of this principle is in nonviral gene and oligonucleotide transfer with the use of chimeric multifunctional biomolecules. These multidomain molecules have individual domains for forming complexes with the DNA, targeting, and evading cellular degradation/ scavenging pathways.47 Chimeric molecules are also used in the vaccine development process for efficient targeting and delivery, in immunotherapeutics to generate therapeutic-friendly antibodies,48,49 in understanding the biophysical properties of molecules, and in identifying domains that contribute to enzyme stability and catalytic activity.50 C. Molecular Bioengineering Technologies Advances in genetic engineering have resulted in the development of new techniques as well as the improvement of existing methods for rapidly and efficiently generating improved biomolecules. A wide range of moleculessenzymes, antibodies, peptides, nucleic acids, and miscellaneous organicssall can be altered at either the gene level or the protein level using techniques such as mutagenesis, directed evolution, and molecular conjugation. In this section, we describe the techniques

Ind. Eng. Chem. Res., Vol. 41, No. 3, 2002 447 Table 2. Modifications of Biological Molecules and Considerations for Their Selection modification technique

chemical or genetic

need DNA sequence?

site-directed mutagenesis

genetic

yes

random mutagenesis directed evolution phage display

genetic genetic genetic

no yes yes

combinatorial biosynthesis biological chimeras combinatorial chemistry

genetic genetic chemical

yes yes no

chemical chimeras structure-based drug design

chemical chemical

yes no

used in molecular bioengineering, broadly classified as either genetic or chemical in origin. Table 2 summarizes some of the considerations involved in choosing a molecular modification for a particular problem. C.1. Molecular Bioengineering using Genetic Technologies. C.1.1. Mutagenesis. The basic approach to enhancing protein function involves altering the DNA that is used as the template for synthesizing the protein, so that the properties of the expressed protein are also altered. Mutagenesis of DNA (i.e., altering of the sequence of bases in the DNA molecule) is a common means of introducing changes in the molecule and can be based on rational design principles. When this approach is applied to specifically modify certain bases in the gene, it is termed site-directed mutagenesis (SDM). The information required for making precise amino acid changes is derived from the protein structure, mechanism of action, or function, which are, in turn, based on molecular modeling, X-ray crystallography data, or comprehensive biochemical characterization. SDM represents a powerful tool for understanding function-sequence relationships in proteins.51,52 For example, SDM has been used with knowledge of enzyme structure, interactions with various ligands, and mechanism of action to increase the catalytic activity of enzymes. This approach has been successfully applied to create improved enzyme variants for superoxide dismutase and isocitrate dehydrogenase.53 However, the structural and mechanistic information needed for SDM to result in an effective modification is often lacking; hence, the application of rational design approaches to the engineering of industrially important enzymes has been limited.54,55 Furthermore, protein function is not solely determined by a few amino acids, and successful enzyme variants often require multiple mutations in different regions of the protein. In fact, successful variants generated by other methods such as random mutagenesis often show mutations in regions that are not predicted by molecular modeling to be important for enzymatic activity.56,57 A possible explanation for this observation could be that proteins have evolved to be locally optimum, so that global changes (changes in multiple domains) are needed to observe any further improvement. When the goal of mutagenizing a molecule is to enhance the baseline value function of a protein rather than to add a new function to the protein,58,59 a more random approach can be adopted. Using this strategy, multiple mutations are introduced in the gene encoding the protein, which might exert a synergistic effect on protein function that is greater than that from single mutations. This has been corroborated by Hirano et al.,

need protein structure? no, but structural information can be helpful no no no, but structural information can be helpful no no no, but structural information can be helpful no yes

obtain library of variants? no yes yes yes yes no yes no no

who observed a cumulative 40-fold increase in the activity of the enzyme ribonuclease H1 from three individual mutations.60 Because no prior information on protein structure or function is required for random mutagenesis, it can potentially be applied to virtually any protein. The vast majority of random mutants is unaffected or exhibits a decrease in activity or function. Therefore, the number of protein variants generated by random mutagenesis is generally quite large, and a robust screening method is required to identify the variants with desired properties. Current opinion is that a combination of rational (to predict regions in the protein that might be worth modifying) and random (to generate a vast library of mutants in a predicted region) mutagenesis represents the most attractive route for engineering proteins. This has been demonstrated by researchers in “converting” the E. coli pyruvate oxidase enzyme to R-ketobutyrate oxidase by randomly changing the amino acid residues in a model-predicted localized region.61 Mutagenesis has been successfully applied to bioprocessing and pharmaceutical applications, where the focus is on increasing the yield of the desired product rather than improving its functional properties. A striking example of this approach is the engineering of a Rhodococcus strain to produce two pharmaceutical intermediates, cis-(1S-,2R)-indandiol and trans-(1R,2R)-indandiol.62,63 Using information from metabolic flux analysis of the indene bioconversion pathway, pathways that led to unwanted byproducts as well as to modified enzyme activities were eliminated by mutagenesis, resulting in a more selective transformation to cis-indandiol. C.1.2. Directed Evolution. Directed evolution, also called DNA shuffling and molecular evolution, improves upon random mutagenesis by incorporating recombination of different mutated gene fragments.64,65 A single gene or a family of related genes is randomly fragmented, and the population of random fragments amplified using multiple cycles of the polymerase chain reaction (PCR), with the random fragments themselves priming the PCR reaction. As the number of PCR cycles increases, the length of the random fragments also increases, and eventually, one of the random fragments accidentally primes a mutant fragment, resulting in amplification of the mutant fragment. This mixing during PCR introduces a few mutations that are then cloned in bacteria and selected for using recombinant DNA methods. In one sense, directed evolution can be called an in vitro Darwinian evolution, as the variants generated during one cycle of PCR are used as the template for the next cycle of evolution.66 Directed evolution has been successfully applied in several

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diverse fields: improving the activity of the industrial enzymes protease, amylase, lipase, cellulase, and xylenase;67 improving the efficiency of bioconversions;68 developing extremophilic enzymes;69,70 enhancing the enantioselectivity of compounds such as L-methionine;71,72 and developing retroviral strains.73 In addition to the evolution of single genes, entire families of homologous genes have been successfully shuffled to improve a range of enzyme properties.74 Ness et al. generated shuffled subtilisin from 26 similar genes and screened for enhancement in four properties (activity at 23 °C, thermostability, solvent stability, and pH dependence) that have previously been targeted by rational engineering and shuffling strategies.75 Using this family shuffling approach, variants having unnatural combinations of desired properties (such as activity at 23 °C and thermostability) were also generated. Furthermore, the most thermostable variant possessed a 20% difference in amino acid composition, compared to the most thermostable parent enzyme, which underscores the advantage of DNA shuffling over site-directed mutagenesis or rational design strategies in generating diversity of both sequence and function. Directed evolution is also being combined with rational design to evolve proteins with improved functional properties. By using comparative analysis of structural and biochemical data, the common R/β-barrel scaffold from the enzyme indole-3-glycerol-phosphate synthase (IGPS) was used to evolve phosphoribosyl anthranilate isomerase (PRAI) enzymatic activity, which was then followed by directed evolution to result in a new PRAI that had catalytic activity comparable to the natural enzyme with an even higher specificity.76 Although the potential for directed evolution of enzymes and proteins is vast, the limiting factor in directed evolution is the ability to screen for variants showing improved performance. Each round of directed evolution can result in thousands of mutants, the screening of which can be expensive and time-consuming. The extent to which directed evolution will be applied to improve the properties of industrially relevant proteins will depend to a great degree on the effectiveness of high-throughput screening methods. C.1.3. Phage Display. Phage display77,78 is a tool that exploits libraries of proteins, expressed on the surface of filamentous bacteriophage, to detect target molecules.77,78 The peptides are physically linked to the genetic information that encodes them, providing a means to rapidly isolate DNA clones corresponding to selected peptides. For this reason, phage display has found application in the optimization of pharmaceutical candidate molecules.79 Although phage display is primarily used as a high-throughput screening method (and is discussed in that context elsewhere in this review), it can also be used as a method for modifying proteins and generating diversity. Smith80 has described a variation of phage display known as patch engineering, in which amino acid residues are randomized simultaneously in several noncontiguous regions of the protein and displayed on the phage surface. When the protein is folded, the three-dimensional structure results in the formation of a contiguous patch in the folded protein. With sufficient structural and functional data, this process can be performed such that the folded structure of the protein is unaltered and the protein serves as a support scaffold for surface display of patches that have novel binding activities. Using patch

engineering, the binding affinity of protein Z has been engineered to expand its binding range to include Taq polymerase, human insulin, and apolipoprotein A-1.81 C.1.4. Combinatorial Biosynthesis. Conventional methods of producing new drugs in microorganisms typically involve mutagenesis to inactivate selected genes or the use of recombinant DNA methods to overexpress several genes or pathways involved in the synthesis.82 Using the latter approach, it is now possible to generate novel antibiotic compounds by recombining the naturally occurring biosynthesis pathways. The combinatorial manipulation of different pathways involved in antibiotic synthesis has resulted in the biosynthesis of a library of novel antibiotic compounds having improved properties.15,82 For example, if there are 4 biosynthetic pathways and each pathway contains 4 antibiotic genes, one can generate a library of 256 recombinant microorganisms, each containing a gene for a unique antibiotic. This antibiotic library can then be screened to identify lead candidates for drug development.15 This combinatorial approach has recently been shown to be useful for the production of the antibiotic cancer chemotherapeutic, epirubicin.83 This approach is likely to become even more powerful as functional genomics provides greater understanding of the structure and properties of metabolic pathways. Apart from producing hybrid antibiotics with novel activities, combinatorial biosynthesis can also be used for the production of stereochemically correct pharmaceutical intermediates as well as for the biological modification of antibiotic intermediates that are difficult to modify by chemical methods.84 However, one of the potential problems with producing novel compounds in microorganisms is the difficulty of ensuring that the microorganism is resistant to the novel antibiotic that it is producing.82 It has been speculated that hybrid molecules might not be efficiently secreted from the cell and might eventually act on the host cell itself. Therefore, a great deal of effort is focused on identifying the mechanism(s) of resistance to antibiotics and using this information to generate novel polyketides and peptides. C.1.5. Biological Conjugates and Chimeras. Monoclonal antibodies (Mabs, developed in rodents) have great potential to be used as therapeutic molecules; however, their actual application has been considerably hampered by the immune response they induce upon administration in humans. Therefore, Mabs have been extensively re-engineered in efforts to overcome this limitation. One approach in antibody engineering is to generate conjugates between rodent and human antibodies (chimeric antibodies) so that the immune response generated by the molecule in humans is reduced.49 To generate chimeric antibodies, the genes encoding the variable region of the rodent antibody are cloned into a plasmid vector that also contains the genes for the constant regions of the human antibody. The ensuing chimera has the same antigen binding capacity as the rodent antibody as well as significantly less immunogenic potential. This approach has been further refined based on the knowledge that only certain antigen (the molecule specifically recognized by the antibody) binding loops are required for binding; hence, if only those loops from the rodent Mabs are used for a generating chimeric Mabs, the resulting humanized antibody is likely to be less immunogenic than either the rodent Mab or a chimeric Mab.85,86


Although several chimeric and humanized antibodies have been developed against diseases including nonHodgkin’s lymphoma, rheumatoid arthritis, and breast cancer,87 humanized antibodies have not yet yielded full clinical benefits, as nearly 63% of the patients in the clinical trials for the first humanized antibody, CAMPATH-1H, developed immune responses.49,88-90 The results with CAMPATH-1H highlight the incomplete understanding of sequence-function relationships in both variable and constant regions of antibodies and underscore the challenges of molecular engineering in the therapeutic realm. Nonetheless, recent advances in high-throughput screening such as phage display90 and ribosome display91 will facilitate the development of effective and nonimmunogenic therapeutic Mabs. Protein engineering of antibodies has led to the generation of high-affinity antibodies that have been reduced to the minimum size and binding fragments required.92,93 In particular, the single-chain variable fragment (scFv) molecule has been the focus of recent attention.94 The antigen-binding affinity of scFv’s can be comparable to that of the parent Mab; furthermore, their small size has facilitated the cloning, expression, and overproduction of scFv’s as heterologous proteins in bacteria.86 Recombinant DNA technology has also been used to generate multiple specificities in antibodies (bispecific antibodies or BsAb’s) and multimeric scFv’s (diabodies and triabodies).95,96 Recombinant proteins can also be genetically modified to impart useful functionalities. For example, a sequence can be inserted that encodes a series of histidine residues, which acts as a tag for purification of the protein by immobilized metal affinity chromatography.97 This process has been further developed through the addition (fusion) of gene segments encoding fluorescent markers for facile detection and enzymatic cleavage sites for subsequent removal of the histidine tag from the recombinant protein.98 Entire proteins can also be fused to produce chimeras of molecules that would normally have to associate noncovalently for biological activity. The proteins could be identical, as in the linkage of arc repressor monomers to form a functionally active dimer with an effective dissociation constant that is 2 orders of magnitude less than that of the monomer.45 They can also be formed from two subunits of a ternary complex, as in the fusion of interleukin-6 (IL6) with the gp80 receptor subunit, which results in a 100- to 1000-fold reduction in the dose of IL-6 required for its use in ex vivo hematopoietic progenitor cell expansion.99 Peptides (short oligomers of amino acids with extremely flexible structures) are also under development as vaccines, therapeutics, and antimicrobial agents.100,101 The small size of these molecules has posed problems for their use in vivo, leading to rapid clearance and weak immune response (a strong immune response is needed for vaccines). Several approaches based on molecular conjugates and chimeras have been used to overcome this barrier for various applications. Peptides have been conjugated to larger bacterial proteins and toxins to increase their size and generate a sufficient immune reaction. Several peptides have also been presented on an oligolysine core to generate a multifunctional vaccine.48 Peptide-based probes (peptidomimetics) have been used in the drug discovery process for characterizing receptor-binding candidate molecules.102 In such ap-

plications, it is desirable to generate compounds that show increased affinity and avidity in receptor binding, which can be achieved by reducing the degree of flexibility of the peptides and simulating the structural constraints encountered during their interaction with ligands.103 High-efficiency chimeric antimicrobial peptides, which incorporate the consensus sequences from different antimicrobial peptides, can also constructed by recombinant DNA technology. Using this approach, physical properties of antimicrobial peptides such as surface charge,104 salt tolerance,105 and protease resistance106 have been rationally modified using sequence analysis to improve their therapeutic properties. C.2. Molecular Bioengineering Using Chemical Technologies. C.2.1. Combinatorial Chemistry. The standard procedure for drug development in the pharmaceutical industry is to screen libraries of molecules to select a few lead candidates and, subsequently, to modify these candidates by chemical methods to improve their pharmacological properties.107 Currently, the problem with this approach is two-fold: insufficient numbers of molecules are being generated, and the screening process is not efficient enough to justify the high costs. A solution to the first part of this problem is to use combinatorial synthesis methods to assemble many or all possible combinations of the chemical building blocks present in the base molecule to generate a library of candidate molecules.107-109 The aim of such combinatorial methods is to use automated synthesis to generate libraries efficiently and cost-effectively. Nucleotides,110,111 amino acids,112,113 sugars and carbohydrates,114,115 and polymers116 have all been used in the combinatorial generation of libraries. Such a combinatorial approach is especially useful in catalysis where single- and multistep reactions by cells and enzymes are accelerated to generate libraries of natural products. In other words, combinatorial biocatalysis aims to mimic the evolutionary process of natural product diversification, but generates a significantly greater range of products in much shorter time scales.107,117 Biocatalytic reactions such as the introduction of functional groups (e.g., halogenation and hydroxylation), the modification of existing functional groups (e.g., oxidation and reduction), and the addition of functional groups (e.g., glycosylation and phosporylation) have been used to generate lead molecules for drug discovery from nucleosides, flavonoids, and polyketides.107,109 For example, two rounds of combinatorial biocatalysis generated 600 derivatives of the flavonoid bergenin, which were then screened for activity against urokinase and xanthine oxidase to choose improved derivatives of bergenin. As with any other modification scheme (directed evolution, combinatorial biosynthesis) that generates many candidate molecules, the usefulness of combinatorial chemistry is limited by the restrictions on the number of molecules that can be screened in a cost-effective manner. C.2.2. Molecular Conjugates and Directed Chemical Modifications. Existing biomolecules, including nucleic acids118 and proteins,119 can be modified chemically to improve functional properties. With nucleic acids, the most frequent type of conjugation is the complexation of oligonucleotide or plasmid DNA to cationic lipids to increase delivery for antisense oligonucleotide therapeutics and nonviral gene transfer.120 Alternatively, ligands specific for receptors expressed preferentially on a target cell type have been used, often in conjunction

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with cationic moieties such as poly-L-lysine, to enhance the magnitude and specificity of delivery for both oligonucleotides and plasmids.121-125 Some investigation is now underway toward intracellular targeting of nucleic acids, such as through conjugation of nuclear localization signal peptides to direct nonviral gene transfer vectors to the cell nucleus.126 Dextrans, poly-L-glutamic acid (PLGA), and heparin oligomers have been conjugated to proteins to enhance their pharmacological properties and tissue-specific delivery in various applications.127-129 Dextrans are glucose polymers that are conjugated (directly or using a spacer molecule) to proteins and drugs to improve their pharmacokinetic and pharmacodynamic properties.130,131 Because the biodistribution and elimination of dextran-conjugated molecules is dependent on the charge and molecular weight of the polymer conjugated, dextran molecules can be incorporated rationally into conjugates for specific applications. Another application of molecular conjugates is in targeting of drugs; for example, conjugation of galactosylated PLGA (via an ethylenediamine spacer or hydrazone bond) to the drug prostaglandin resulted in efficient delivery to hepatocytes in vivo.132 Conjugates of dextran and poly(ethylene glycol) have also been used to generate microparticles that are more stable and exhibit improved surface properties.133 Hydrophilic targeting moieties are then attached to the surface to increase tissue-specific delivery of conjugated drugs. Genetic and chemical modification methods have also been combined for several applications. A cysteine thiol site has been genetically inserted near the recognition site of streptavidin, which was then used to chemically conjugate low-molecular-weight polymers to streptavidin for potential applications in diagnostics, bioseparations, and biosensors.134 Similarly, chemical conjugation techniques have also been used to generate heterodimers by linking two Fv molecules together, and this approach provides an easier alternative to hybridomas for the cross-linking of antibodies.135 D. Selection and Evaluation of Molecular-Bioengineered Products Even with an increasing repertoire of techniques for manipulating biomolecules, the screening and selection process is critical to identifying which species is optimal for a particular application. In any molecular bioengineering technique, only a small fraction of molecules acquires the desired property or function; hence, identifying and isolating a desired molecule among the unmodified molecules is nontrivial. Depending on the type of molecule being redesigned and the nature of the application, various types of assays are used to screen libraries of molecules for desired variants. Current techniques are also geared toward the generation of libraries of variants, and this has resulted in a major thrust toward adapting conventional biochemical and pharmacological assays to handle large variant libraries. Conventional methods of screening enzyme variant libraries depend on biological selection (based on antibiotic selection pressure) or solid-phase screening.136 Phenotypic selections based on selection pressure are proving to be less effective, as random mutations can result in growth without an increase in specific enzyme activity. In addition, solid-phase formats are not available for many screening assays. Thus, there is considerable focus on developing screening methods that can be

adapted to a high-throughput format for screening large libraries. This section focuses on some of these screening and selection technologies, with an emphasis on drug discovery applications. D.1. Binding Assays. Screening for protein variants is generally considered to be much easier than screeening for nucleic acid variants because of the ability to screen molecules based on protein activity. However, binding assays have also been applied to the screening of both nucleic acid and protein variants. For example, for the selection of antisense oligonucleotides with high affinity for their target mRNA, a combinatorial screening method was developed based on microarray technology.137 Thousands of antisense oligonucleotides, each complementary to a difference sequence on the target mRNA, were synthesized in predefined locations on a glass surface, which was then exposed to a labeled mRNA of interest. Some debate still exists as to whether in vitro affinity is a reliable indicator of antisense effectiveness in cells or animals, but there is some evidence to support this correlation in cell culture.138 Binding affinity assays are frequently utilized as screens in applications where antibody-antigen or ligand-receptor interactions are of interest.17,139 Highthroughput functional screening assays for receptormediated processes using Saccharomyces cerevisiae have also been developed, which utilize the binding of either receptor agonists or antagonists to allow evaluation of the receptor.140 Another binding-based assay utilizes the interaction between transiently expressed receptors in mammalian cells and compounds that bind to the receptor.141 A β-galactosidase reporter gene is also amplified during cell proliferation, which helps to identify agonist and antagonist molecules that interact with the receptor.142 D.2. Pharmacological Assays. A crucial step in drug development is the assessment of the pharmacological and toxicological characteristics of the engineered molecule. The costs associated with developing a new drug are extremely high, as molecules are rejected at each step of the discovery process on the basis of inappropriate pharmacokinetics and/or toxicity.143,144 Hence, pharmacological assays are used to screen drug candidate molecules and other engineered molecules for these properties. The absorption, distribution, metabolism, and excretion (ADME) kinetics of therapeutic molecules are first determined in cell culture prior to expensive and difficult in vivo studies.143 The value of in vitro screens is not necessarily in replicating an in vivo situation but in eliminating molecules that do not exhibit the desired properties to be developed into a drug. Only candidate molecules that are selected by ADME screens in vitro are subject to in vivo screens using animal models (mainly rodents) of disease or injury. Apart from ADME studies, different routes of administration and biodistribution (e.g., distribution of antisense oligonucleotides or other drug molecules in various tissues upon administration) are also determined.145,146 The toxicological profile (physiological and immunological changes) after administration of the test molecule is obtained in vivo to assess the overall efficacy of the biomolecule in a specific function. Although these methods are typically low-throughput, the power of these methods is being improved by the use of predictive models to determine pharmacokinetic outcomes.143 D.3. High-Throughput Assays. In high-throughput screening (HTS), conventional assays are automated


and/or miniaturized to screen as many novel or modified compounds as possible while keeping reagent costs to a minimum.140 The simplest form of cell-free HTS aims to adapt conventional 96-well (and newer, higherdensity) assay formats to use nonradioactive detection assays such as chemiluminescence and fluorescence. Several types of fluorescence assays have been developed for screening chemical libraries, including fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), and homogeneous time-resolved fluorescence (HTRF), all of which simplify the labeling process as well as increase the sensitivity of the screening process.140,147 Harris et al.148 have constructed soluble positional protease libraries of 137, 180 fluorogenic peptides and screened a wide range of proteases, including the serine proteases papain, trypsin, and chymotrypsin. These profiles allow the design of sensitive and selective substrates and inhibitors for proteases. In addition to fluorescence, chemiluminescence has been used frequently in enzyme-linked immunosorbent assays (ELISA) for screening horseradish peroxidase and alkaline phosphatase-conjugated biomolecules such as antibodies. Cell-based screening systems are emerging as potential methods for screening libraries of protein variants or enzymes based on their functional properties. These assays utilize mutants of fluorescent reporter probes, such as green fluorescent protein (GFP) and luciferase, in combination with resonance energy transfer to screen biomolecules.149,150 With advances in microfabrication and microfluidic processing techniques, these probes might eventually be incorporated into “lab-on-a-chip” devices.151 D.4. Phage Display. One of the most powerful technologies for screening protein variants based on protein-ligand interactions is the phage display method. Display methods are available in bacteria152,153 and yeast,154 and libraries in these systems can contain up to 109 different molecules. Phage and ribosome display enables the screening of at least 1013 members per library and, hence, increases the chances of finding a desired variant in the library.155 Phage-display-based screening has resulted in the selection of inhibitor molecules to specific enzymes, receptors, and protein purification agents.79 By combining phage infectivity with function, phage display has also been used to screen for catalytic antibodies, peptide targets for proteases, and protease-resistant proteins.156 Because of the specific nature of protein-ligand interactions, phage display only selects for correctly folded variants of a library; however, it does not provide any further information on the relative affinities among the selected variants.157 Apart from protein-ligand interactions, other biophysical properties such as stability at elevated temperatures and folding have been used as selection criteria for phage display. Ruan et al. showed that it is possible to select thermostable mutants from a library of subtilisin variants with three rounds of phage display.158 Highly stable variants of barnase have also been selected by taking advantage of the increased number of proteolytic cleavage sites exposed in unfolded protein variants compared to correctly folded ones, thereby combining phage infectivity with proteolytic sensitivity.159 A recent supplement to the phage display method in screening libraries is the use of flow cytometry, in which

a fluorescently labeled ligand is used to screen a library, with the cells expressing the library subsequently sorted on the basis of their fluorescence levels. Flow cytometry can be used for both equilibrium and kinetic discrimination of ligand-receptor interactions, in the latter case abetted by the use of a competitive inhibitor.160 The main limitation of flow cytometry applied to enzymes is the requirement for the substrate to be internalized by the cell and the fluorescent product retained inside the cell. The display of enzyme libraries on the surface of cells overcomes these limitations and helps to adapt flow cytometry for high-throughput screening applications. Olsen et al. have used protein surface display coupled with flow cytometry and fluorescence resonance energy transfer (FACS/FRET) to isolate a mutant protease that demonstrated a 60-fold increase in catalytic activity. This new approach uses the activity of the molecule as the basis of screening instead of ligand binding.161 This combination of display techniques based on binding and a sensitive analytical technique based on activity can be adapted for quantitative, highthroughput screening of vast enzyme libraries.136 D.5. Microarrays. The advent of microarray technology has made it feasible to spot thousands of oligonucleotides or cDNA species on a glass slide for the analysis and quantification of global changes in gene expression. Microarray analysis of gene expression has great potential to be used effectively in the drug discovery process and in diagnostic applications.162-164 The biggest benefit of microarrays in drug discovery is the ability to simultaneously analyze the effect of lead compound administration on many genes (in principle, the whole genome) in a single experiment. Thus, it is possible to rapidly determine the effect of a modified compound on gene expression in both a target and other tissues in an animal model. The expression profile of different tissues due to the administration of the lead compound can also be compared with those of known toxins to determine whether the lead compound has any potential toxic effects.162 Thus, microarray-based screening and selection can prove to be invaluable in the drug discovery process. The microarray-based approach has been effectively combined with traditional chemical synthesis in the drug discovery process to identify potent kinase inhibitors in yeast.165 Apart from screening lead molecules, microarrays can also be used as a selection method for choosing potential targets against which libraries can be generated and screened. Gene expression profiling can provide insights into biological pathways and networks and the disease states that affect the pathways adversely.166,167 This knowledge can be integrated with gene expression databases to generate targets against which libraries of molecules can be synthesized.163 Summary and Future Directions Recent advances in the sequencing of many bacterial and animal genomes have resulted in a data explosion that presents unprecedented opportunities to improve the performance of existing biomolecules as well as to generate novel molecules for diagnostics, therapeutics, and biotechnology. As this information is rapidly being integrated into research and industrial applications and will eventually further our understanding of molecular and cellular physiology, an interdisciplinary approach, which we term molecular bioengineering, is evolving for the solution of problems related to biomolecules. Already

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a number of examples exist in which unfavorable properties of a candidate biomolecule have been altered to impart more favorable ones. Concurrently, tremendous progress has been made in the development of new methods for generating diversity in biomolecules and efficiently identifying molecules with the desired properties for specific applications. However, many technical challenges still remain to be overcome before the full benefit of molecular bioengineering approaches to biological processes can be realized. Advances in structural proteomics and computational modeling might eventually provide the principles for correlating gene sequence with protein structure and function so that rational redesign of proteins is more feasible. The capability to efficiently introduce diversity in molecules as well as to generate bigger libraries of variant molecules is a key area that merits consideration, as is the ability to be able to perform highthroughput analysis of variant libraries. With the recent emergence of bioinformatics as a critical component of drug development, it is likely that much more information will be available to the molecular bioengineer in selecting a suitable modification for a specific function. Eventually, an integrated approach involving functional genomics, structural and computational biology, and instrumentation will be utilized for advancing the evolution of biomolecules and the improvement of biological processes. It might become possible to tailor designer biomolecules to a particular application by selecting a base molecule (e.g., protein, nucleic acid, small organic) and a series of modular modifications that are optimized on the basis of extensive database information to provide maximal effectiveness with minimal side effects. Clearly, this point remains well in the future, but it represents a goal to fulfill the potential of molecular bioengineering. Acknowledgment The authors thank Patrick Walton and Christina Chan for helpful suggestions and discussions and Sundar Madihally for help in figure preparation. The authors’ work in this area is partially funded by The Whitaker Foundation and the Shriners Hospitals for Children. Literature Cited (1) Monia, B. P.; Johnston, J. F.; Sasmor, H.; Cummins, L. L. Nuclease resistance and antisense activity of modified oligonucleotides targeted to Ha-ras. J. Biol. Chem. 1996, 271, 14533-14540. (2) Manoharan, M. 2′-carbohydrate modifications in antisense oligonucleotide therapy: Importance of conformation, configuration and conjugation. Biochim. Biophys. Acta 1999, 1489, 117130. (3) Walton, S. P.; Roth, C. M.; Yarmush, M. L. In The Biomedical Engineering Handbook; Bronzino, J. D., Ed.; CRC Press: Boca Raton, FL, 2000; Vol. II. (4) Letsinger, R. L.; Elghanian, R.; Viswanadham, G.; Mirkin, C. A. Use of a steroid cyclic disulfide anchor in constructing gold nanoparticle-oligonucleotide conjugates. Bioconjug. Chem. 2000, 11, 289-291. (5) Garrett, S. W.; Davies, O. R.; Milroy, D. A.; Wood, P. J.; Pouton, C. W.; Threadgill, M. D. Synthesis and characterisation of polyamine-poly(ethylene glycol) constructs for DNA binding and gene delivery. Bioorg. Med. Chem. 2000, 8, 1779-1797. (6) Hellgren, I.; Drvota, V.; Pieper, R.; Enoksson, S.; Blomberg, P.; Islam, K. B.; Sylven, C. Highly efficient cell-mediated gene transfer using nonviral vectors and FuGene6: in vitro and in vivo studies. Cell. Mol. Life Sci. 2000, 57, 1326-1333. (7) Matoba, N.; Doyama, N.; Y, Y.; Maruyama, N.; Utsumi, S.; Yoshikawa, M. Design and production of genetically modified

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Received for review March 19, 2001 Revised manuscript received July 13, 2001 Accepted July 18, 2001 IE0102549

Molecular Bioengineering - Industrial & Engineering Chemistry

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