Genetically Encoded Synthesis of Protein-Based Polymers with

Biomacromolecules 2002, 3, 357-367

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Genetically Encoded Synthesis of Protein-Based Polymers with Precisely Specified Molecular Weight and Sequence by Recursive Directional Ligation: Examples from the Elastin-like Polypeptide System Dan E. Meyer and Ashutosh Chilkoti* Department of Biomedical Engineering, Box 90281, Duke University, Durham, North Carolina 27708-0281

Biomacromolecules 2002.3:357-367. Downloaded from pubs.acs.org by OPEN UNIV OF HONG KONG on 01/23/19. For personal use only.

Received October 22, 2001; Revised Manuscript Received December 19, 2001

We report a new strategy for the synthesis of genes encoding repetitive, protein-based polymers of specified sequence, chain length, and architecture. In this stepwise approach, which we term “recursive directional ligation” (RDL), short gene segments are seamlessly combined in tandem using recombinant DNA techniques. The resulting larger genes can then be recursively combined until a gene of a desired length is obtained. This approach is modular and can be used to combine genes encoding different polypeptide sequences. We used this method to synthesize three different libraries of elastin-like polypeptides (ELPs); each library encodes a unique ELP sequence with systematically varied molecular weights. We also combined two of these sequences to produce a block copolymer. Because the thermal properties of ELPs depend on their sequence and chain length, the synthesis of these polypeptides provides an example of the importance of precise control over these parameters that is afforded by RDL. Introduction Protein-based polymers, which are composed of repeat units of natural or unnatural amino acids, have recently emerged as a promising new class of materials.1-4 They are attractive from a fundamental materials science perspective because their genetically encoded synthesis provides precise control, to a level unattainable using chemical polymerization techniques, over the primary architectural features of polymers, namely sequence, chain length, and stereochemistry. Furthermore, these materials frequently also have desirable mechanical, chemical, and biological properties (e.g., biocompatibility, biodegradation) that makes their use appealing as biomaterials and tissue engineering scaffolds. The sequence and molecular weight (MW) of repetitive polypeptides are of particular importance because these two primary architectural variables determine the physicochemical properties of the macromolecule. These variables are also important for in vivo applications because the MW controls pharmacokinetics and transport phenomena, while the amino acid sequence can impart biological activity and often determines the biodegradation of the polypeptide. The precise and rapid synthesis of genes encoding a polypeptide of desired sequence and length is therefore a key requirement for producing genetically encoded, repetitive polypeptides for specific applications. Although a number of different strategies have been developed to assemble synthetic genes for such polypeptides,5-7 most methods have focused upon the simultaneous generation of a library of oligomeric genes by concatemerization of a monomer gene.8-12 Concatemerizationsthe self-ligation of a DNA * To whom correspondence should be addressed at [email protected].

monomer with cohesive endsshas the advantage of creating, in a single ligation step, a library of genes that encode oligomeric polypeptides with the same repeat sequence but different sizes. Although concatemerization is rapid, it sacrifices precise control over the oligomerization process because it is a statistical process that yields a population of DNA oligomers with a distribution of different lengths. The average degree of oligomerization can be partially controlled by varying the ligation conditions, but concatemerization does not guarantee the synthesis of a gene with a desired length. Because concatemerization does not guarantee a priori that a clone will be isolated that contains an insert encoding the desired number of peptide repeats, it is a useful synthetic strategy when a range of MWs need to be rapidly generated but where synthesis of a gene encoding a specified number of peptide repeats is of secondary importance. We report here a general strategy, which we term “recursive directional ligation” (RDL), for the synthesis of repetitive polypeptides of a specified chain length. This genelevel approach involves controlled, stepwise oligomerization of a DNA monomer to yield a library of oligomers ranging from the monomer to an oligomer of a required chain length (Figure 1). We employed RDL to synthesize elastin-like polypeptides (ELPs), which are based on the repetitive pentapeptide motif Val-Pro-Gly-Xaa-Gly (where the “guest residue” Xaa is any amino acid except Pro). ELPs are an interesting class of polypeptides because they undergo an inverse temperature phase transition.13,14 ELPs are soluble in aqueous solution below the inverse transition temperature (Tt, also known as the lower critical solution temperature or LCST). However, when the temperature is raised above the Tt, they undergo a sharp (∼2 °C range) phase transition

10.1021/bm015630n CCC: $22.00 © 2002 American Chemical Society Published on Web 01/30/2002

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Figure 1. Overview of gene oligomerization by RDL. A monomer DNA segment encoding a polypeptide sequence of interest is seamlessly self-ligated. The process is repeated, doubling the gene length with each step, until the gene of a desired length is obtained (left pathway). Other genes encoding different polypeptide sequences can be incorporated at any step (right pathway). In this example, a gene encoding a different sequence is ligated to the first sequence to produce a diblock copolymer, which in turn can be further oligomerized to create more complex block copolymers.

leading to desolvation and aggregation of the polypeptide. The transition can be induced by changes in temperature, ionic strength, or pH and is completely reversible.14 Numerous applications of ELPs in biotechnology and medicine have been proposed.7,15-20 We developed RDL as a synthetic strategy for ELPs in particular because we wished to study the effect of sequence, MW, and the architecture (e.g., in ELP block copolymers) of ELPs on their thermal behavior in solution. The ability to precisely control these variables is important because the Tt of a given ELP is independently related to its sequence (i.e., the identity of the guest residues) and to its chain length.21,22 Using RDL, we synthesized genes encoding oligomeric libraries of three different ELP sequences and combined two of these genes to encode a block copolymer. Because the sequence and chain length determine the thermal properties of ELPs, these polypeptides provide an excellent example of the importance of the precise control over these parameters that can be obtained by RDL. Materials and Methods Materials. Restriction endonucleases, the pUC19 cloning vector, and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA). Calf intestinal alkaline phosphatase (CIP) and Taq DNA polymerase were obtained from Gibco BRL-Life Technologies (Grand Island, NY). Plasmid DNA was purified using spin miniprep kits from QIAGEN, Inc. (Valencia, CA). The pET-25b(+) expression vector and the BLR(DE3) E. coli strain were purchased from Novagen Inc. (Milwaukee, WI). All cultures were grown in CircleGrow medium from Q-BIOgene (Carlsbad, CA), and polypeptide expression was induced with isopropyl β-Dthiogalactopyranoside (IPTG) from Teknova, Inc. (Half Moon Bay, CA). Custom oligonucleotides were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). Precast Mini-Protean SDS-PAGE gels were from BioRad, Inc. (Hercules, CA). Nomenclature. We distinguish the different ELP constructs using the notation ELP[XiYjZk-n]. The bracketed

Meyer and Chilkoti

capital letters are the single letter amino acid codes specifying the guest residues in the ELP sequence, and the subscripts designate the number of Val-Pro-Gly-Xaa-Gly repeats for each corresponding guest residue in the monomer gene. The total length of the ELP gene in number of pentapeptides is specified by n. For example, ELP[V5A2G3-180] is an ELP of 180 pentapeptides in length that has a repeat unit composed of 10 pentapeptides with the guest residues Val, Ala, and Gly in a 5:2:3 ratio, respectively. If n is unspecified, the notation refers to a MW library of the given ELP sequence, rather than to an ELP construct of specific length. Monomer Gene Synthesis. Standard molecular biology protocols were used for DNA manipulation, E. coli culture, and protein expression.23 The DNA sequences of the monomer genes for the three libraries are shown in Figure 2A. For the ELP[V5A2G3] library, a synthetic gene (150 bp) encoding a 50 amino acid ELP repeat was constructed from four 5′-phosphorylated, PAGE-purified synthetic oligonucleotides. Although this is the monomer gene for RDL, we term it a “10-mer” because it encodes 10 pentapeptides. The oligonucleotides were annealed by heating an equimolar mixture of the four oligonucleotides (2 µM each in ligase buffer) to >95 °C and then slowly cooling to room temperature to form a double-stranded DNA cassette with EcoR I and HinD III compatible ends. pUC19 was codigested with EcoR I and HinD III and enzymatically dephosphorylated using CIP. The linearized pUC19 vector was then purified using a microcentrifuge spin column purification kit and eluted in sterile, deionized water. The annealed oligonucleotides were then ligated to the linearized vector (∼200 U ligase, ∼0.1 pmol vector, and ∼1 pmol insert incubated in 20 µL ligase buffer at 16 °C for 2 h). A 10 µL portion of the ligation mixture was combined with 100 µL of chemically competent E. coli cells (XL1-Blue strain), and the cells were transformed by heat shock (30 min 4 °C, 60 s 42 °C, 5 min 4 °C), spread on CircleGrow medium agar plates supplemented with ampicillin (100 µg/mL), and incubated overnight at 37 °C. Colonies were initially screened by blue-white screening and subsequently verified by agarose gel electrophoresis of colony PCR products. The DNA sequence of putative inserts was further verified by dye terminator DNA sequencing (ABI 370 DNA sequencer). The ELP[V5-5] and ELP[V1A8G7-16] monomer genes were constructed similarly (Figure 2A). Gene Oligomerization. One round of RDL oligomerization of the ELP[V5A2G3-10] gene is described here as an example. Additional rounds proceed identically, except that the products of previous rounds serve as the starting materials. After the ELP[V5A2G3-10] monomer gene was constructed, a ELP[V5A2G3-20] gene was synthesized by ligating the 10-mer insert into a vector containing the same 10-mer gene, as follows. The vector, containing a copy of the gene of the monomer ELP[V5A2G3-10], was linearized with PflMI (∼10× overdigestion for 6 h), enzymatically dephosphorylated with CIP, and then purified using a microcentrifuge spin column purification kit. A separate sample of vector was doubly digested with PflMI and Bgl I to excise the gene encoding ELP[V5A2G3-10]. After digestion, the reaction products were separated by agarose gel

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Figure 2. Gene and corresponding polypeptide sequences. (A) Monomer genes for the three ELP libraries are shown. The PflM I and Bgl I sites, which are used for RDL, are shown with recognition sequences in bold, cleavage sites indicated by arrows, and cohesive ends underlined. (B) Any ELP gene can be ligated into the Sfi I site of the expression vectors, which also encode short leader and trailer peptides.

electrophoresis, and the insert was purified using a gel extraction microcentrifuge spin column kit. During purification of the insert from the agarose gel slice, vortexing or heating of the samples above 37 °C was avoided to prevent damage to the DNA. The purified insert and the linearized vector were ligated and transformed into XL1-Blue cells using the protocol described above. Transformants were initially screened by colony PCR and/or diagnostic restriction endonuclease (RE) digests and further confirmed by DNA sequencing. Expression Vector Construction. Expression vectors that are compatible with the ELP genes were constructed by modifying the DNA sequence spanning Nde I to EcoR I of pET-25b(+), a commercial T7-lac expression vector, by cassette mutagenesis to incorporate a unique Sfi I recognition site (Figure 2B). The modified pET-25b(+) expression vector was digested with Sfi I (∼10× overdigestion for 6 h), dephosphorylated, and purified using a microcentrifuge spin column kit. The ELP gene was excised from the pUC-19 vector by digestion with PflM I and Bgl I, and the excised ELP gene was purified by agarose gel extraction following gel electrophoresis. The Sfi I linearized pET-25b vector and the ELP-encoding gene were ligated and transformed as

described above, and plasmids isolated from the resulting transformants were screened by diagnostic RE digest and sequenced. Expression. The expression vectors were transformed into the E. coli strain BLR(DE3) for expression. Typically, starter cultures (250 mL flasks containing 50 mL of medium supplemented with 100 µg/mL ampicillin) were inoculated with transformed cells from a fresh agar plate or from DMSO stocks stored at -80 °C, and incubated overnight at 37 °C with shaking (300 rpm). The confluent starter cultures were then centrifuged at 3000g for 15 min at 4 °C to remove β-lactamase, and resuspended in 10 mL of fresh medium. Expression cultures (4 L flasks containing 1 L of medium with 100 µg/mL ampicillin) were inoculated with 2 mL of the resuspended starter culture and incubated with shaking (∼300 rpm) at 37 °C. When the OD600 reached ∼0.8-1.0 (typically about 3.5 h postinoculation), expression was induced by the addition of IPTG to a final concentration of 1 mM. The cells were typically harvested 3 h after induction by centrifugation at 3000g for 20 min at 4 °C, resuspended in 35 mL of cold, low ionic strength buffer (typically PBS: 137 mM NaCl, 2.7 mM KCl, 4.2 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.3), and lysed by sonic disruption at 4 °C (90 s of sonication at maximum power, using 10 s pulses

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separated by 20 s; 550 Sonic Dismembrator, Fisher Scientific, Pittsburgh, PA). The cell lysate was centrifuged at 20000g for 15 min at 4 °C to remove insoluble cellular matter. Soluble nucleic acids were precipitated by the addition of polyethylenimine (0.5% final concentration, w/v) and removed by centrifugation at 20000g for 15 min at 4 °C. ELP Purification. ELPs were purified by inverse transition cycling.10,17 Briefly, the ELPs were selectively aggregated by heating the cell lysate (typically 30-45 °C) and/ or by adding NaCl (typically 0.5-2 M). The aggregated protein was separated from solution by centrifugation at 10000g for 15 min at 30-45 °C. The supernatant, containing soluble contaminants from the lysed E. coli cells, was decanted and discarded. The pellet containing the ELP was resolubilized in cold, low ionic strength buffer. Once fully resuspended in solution, the first round of inverse transition cycling was completed with a final centrifugation step at 15000g, for 10 min at 4 °C to remove any remaining insoluble contaminants. Typically, two rounds of inverse transition cycling (warm centrifugation, pellet resuspension, and subsequent cold centrifugation) were sequentially performed to purify the ELP. Characterization of the Expressed ELPs. The ELPs were characterized by SDS-PAGE, mass spectrometry, and UV-vis spectrophotometry. The concentration of ELP solutions was determined spectrophotometrically using the molar extinction coefficient of Trp at 280 nm (5690 M-1 cm-1). SDS-PAGE gels were visualized by copper staining.10,24 Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was performed by the Duke University Mass Spectrometry Facility in the Department of Chemistry using a PE Biosystems Voyager-DE instrument equipped with a nitrogen laser (337 nm). The MALDI-MS samples were prepared in an aqueous 50% acetonitrile solution containing 0.1% trifluoroacetic acid, using a sinapinic acid matrix. Thermal Characterization. To characterize the ELP inverse temperature transition, the OD350 of ELP solutions (typically 25 µM ELP in PBS) was monitored as a function of temperature on a Cary 300 UV-visible spectrophotometer equipped with a multicell thermoelectric temperature controller (Varian Instruments, Walnut Creek, CA). The heating and cooling rates were 1 °C min-1. The derivative of the turbidity profile with respect to temperature was numerically calculated, and the Tt was defined as the solution temperature at the maximum of the turbidity gradient. The size of ELP aggregates formed during the inverse temperature transition was characterized as a function of temperature by dynamic light scattering (DLS) using a DynaPro-LSR DLS instrument equipped with a Peltier temperature control unit (Protein Solutions, Charlottesville, VA). A 25 µM solution of ELP in PBS was centrifuged at 16000g for 10 min at 4 °C to remove air bubbles and insoluble debris, and the cold supernatant was then filtered through a 20 nm Whatman Anodisc filter. Light scattering data (15 measurements, each with a 5 s acquisition time) were collected at 1 °C intervals as the solution was heated from 35 to 60 °C. The autocorrelation function was analyzed using a regularization algo-

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Figure 3. The molecular biology steps of RDL. (A) A synthetic monomer gene is inserted into a cloning vector. (B) The gene is designed to contain recognition sites for two different restriction endonucleases, RE1 and RE2, at each of the coding sequence. (C) An insert is prepared by digestion of the vector with both RE1 and RE2 and subsequently ligated into the vector that has been linearized by digestion with only RE1. (D) The product contains two head-totail repeats of the original gene, with the RE1 and RE2 sites maintained only at the ends of the gene. (E) Additional rounds of RDL proceed identically, using products from previous rounds as starting materials.

rithm for spherical particles provided by the manufacturer (Dynamics software version 5.26.37). Results and Discussion Overview of RDL. A schematic of RDL is shown in Figure 3. A synthetic oligonucleotide cassette encoding the monomer gene is first ligated into a cloning vector such as pUC19. The oligonucleotides are designed so that EcoR I and HinD III compatible cohesive ends are produced upon annealing, which enables the annealed product to be directly ligated into EcoR I and HinD III cleaved pUC19 (Figure 3A). The monomer gene is designed to encode a defined number of pentapeptide repeats, while incorporating two additional restriction endonuclease recognition sites on each end of the coding sequence, internal to the EcoR I and HinD III sites (Figure 3B). These additional sites, generically labeled RE1 and RE2 in Figure 3, are used to oligomerize the gene by RDL as follows. An insert is produced by cleaving the plasmid that harbors the monomer gene with both RE1 and RE2, and a linearized vector is produced by separately digesting another aliquot of the same plasmid with only RE1 (Figure 3C). The purified insert is ligated into the linearized vector, resulting in dimerization of the gene (Figure 3D). The monomer gene is designed such that gene oligomerization by RDL achieves three goals. First, the insert is ligated with its directionality preserved in a head-to-tail orientation upon ligation into the vector. Second, the ligation


Figure 4. Gene design for RDL. The polypeptide sequence to be oligomerized (A) specifies a degenerate DNA coding sequence (B). Pairs of restriction endonucleases (C & D) are checked for compatibility by combining the 5′ segment of one enzyme recognition sequence with the 3′ segment of the second enzyme (E). (Recognition sequences are in bold, sites of cleavage are marked by vertical arrows, and single-stranded overhangs created by cleavage are underlined.) The degenerate coding sequence is searched for the paired recognition sequence (F), and then the coding sequence is shifted so that the cleavage sites of the two enzymes are located at opposite ends of the gene (G).

is seamless in that extraneous residues are not introduced at the ligation site. Third, the original recognition sites for RE1 and RE2 are maintained at each end of the dimerized gene, but neither recognition site is generated at the internal site of ligation. Therefore, the oligomer assembled in any round of RDL (e.g., dimer in the first round) can be used in future rounds of RDL as the insert and/or the vector (Figure 3E). Later rounds of RDL are identical to the first round, except that products from previous rounds serve as the source of the insert and vector. Selection of RE1 and RE2 for Recursive Directional Ligation. RDL requires the selection of a pair of restriction endonucleases that satisfy four important requirements. First, they must have different recognition sequences so that the DNA can be selectively cleaved either by one or by both of the enzymes and so that neither site is re-formed at the internal site of ligation. Second, the two enzymes must produce complementary, single-stranded DNA overhangs upon cleavage. Third, at least one of the two sites (generically, RE1) should be unique on the cloning vector so that digestion with the enzyme cleaves the plasmid only at a single site. Finally, the recognition sequences of both REs must be compatible with the coding sequence of the polypeptide such that, upon the ligation of two gene segments, the repeat sequence of the polypeptide is not disrupted at the internal site of ligation. To fulfill these four requirements, we have chosen PflM I as RE1 and Bgl I as RE2 for our implementation of RDL for oligomerization of ELP sequences. PflM I and Bgl I have different recognition sequences, and thus the pair meets the first requirement above. Furthermore, both enzymes have a split palindromic recognition sequence located on either side of five unconstrained bases, three of which comprise the 3′ overhang produced on cleavage (Figure 4C,D). In contrast with the more common RE cleavage patterns in which the site of cleavage is located


within a palindromic recognition sequence, the unconstrained overhang sequence produced by these enzymes is useful for two reasons. First, this enables the selection of the same overhang sequence for both enzymes, which for our ELP genes was 5′-GGC-3′. Therefore, the ends created by digestion with PflM I are compatible with those created by digestion with Bgl I, thereby meeting the second requirement above. Second, these overhang bases are nonpalindromic, which forces ligation of the insert in a head-to-tail orientation with respect to the gene segment contained within the vector. The pUC19 cloning vector has no PflM I sites, and therefore the single site introduced upon insertion of the monomeric gene is unique, meeting the third requirement outlined above. This allows the vector to be linearized by digestion with PflM I, to receive the insert. It is also desirable that the second site be unique, but this requirement is less stringent. For example, Bgl I has two recognition sites in pUC-19 in addition to the site that is introduced into the plasmid at the 3′ end of the ELP gene. Therefore, when preparing the ELP gene insert by digestion of its vector with PflM I and Bgl I, four fragments are formed. The desired fragment containing the ELP gene can usually be separated from the other three fragments by extraction of the appropriate band following agarose gel electrophoresis. Furthermore, we selected an overhang sequence for the introduced Bgl I and PflM I sites that is incompatible with the cohesive ends produced by the two sites that are native to pUC19, and therefore the three vector-derived fragments cannot ligate into the linearized vector prepared by digestion with PflM I. Therefore, if an ELP gene insert and one of the contaminating fragments are of similar size and cannot be separated by electrophoresis, it is not critical to purify the insert from all three contaminating bands. It is necessary, however, to purify the insert from at least one of the contaminating bands to minimize background arising from religation of all four fragments of the wild-type vector. The fourth and final requirement relates to the compatibility of these two enzymes with the gene sequence, as illustrated in Figure 4. Recognition sites for RE1 and RE2 must be present at opposite ends of the coding sequence, and the sequences must be designed such that the repetitive polypeptide sequence is not disrupted upon ligation of a RE1cleaved end to a RE2-cleaved end. To design a gene to satisfy these requirements, a potential RE pair is checked for suitability by first combining the 5′ end of one RE’s recognition site, divided at the cleavage point, with the 3′ end of its potential partner (Figure 4C-E). To ensure that the polypeptide repeat sequence is not disrupted at the site of ligation during RDL, this paired sequence, which will be formed at the internal ligation site, is compared for compatibility along the complete length of the degenerate codon sequence that encodes the desired polypeptide repeat (Figure 4F). Finally, the coding sequence is shifted, in our example from (Val-Pro-Gly-Val-Gly) to (Val-Gly-Val-Pro-Gly), so that the recognition sequences of these two REs are positioned at each end of the coding sequence (Figure 4GH). (Note that the second half of each recognition sequence must be incorporated external to each end of the coding sequence, as illustrated in Figure 2A.) Finally, the intervening

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sequence between the two sites (denoted by “...” in Figure 4G-H) can be chosen to encode any desired polypeptide sequence, as long as no new RE1 or RE2 sites are introduced. In our designs, we continue the Val-Pro-Gly-Xaa-Gly motif seamlessly from the PflM I site to the Bgl I site (Figure 2A). In addition to meeting the four RDL requirements for a RE pair, another attractive feature of PflM I and Bgl I is the availability of a small pool of similar REs that also produce three-base, unconstrained 3′ overhangs upon cleavage, including AlwN I, Bsl I, Bst AP I, Dra III, Mwo I, and Sfi I. Because the RE recognition sequence constrains the encoded residues flanking each end of the repetitive polypeptide gene, the variety of recognition sequences within this pool of enzymes provides flexibility in the design of expression vectors that are compatible with RDL-derived genes (discussed below). Furthermore, using these compatible enzymes to synthesize libraries of different polypeptide sequences allows modular combination between libraries to form more complex, multidomain repetitive polypeptides (e.g., block copolymers). Although the existence of a suitable pair of REs for a given polypeptide repeat sequence is not guaranteed, RDL is widely applicable because of the large number of REs with different recognition sequences and cleavage patterns that are commercially available. Furthermore, type IIS REs, which cleave at a defined distance to the side of their recognition sequence, could be used to completely eliminate the sequence compatibility requirement by locating the recognition sequence outside of each end the coding region.25 However, they must still conform to the other RE requirements, and in particular they must produce a cohesive end that is compatible with at least one other RE. There are numerous type IIS REs that fulfill these requirements, and those that produce four-base, unconstrained 5′ overhangs upon cleavage would be particularly well suited to RDL, including Alw26 I, Bbs I, BbV I, BbV II, Bsa I, BsmA I, BsmB I, BsmF I, BspM I, Esp3 I, Fok I, Fin I, and SfaN I. Using type IIS REs for RDL would provide the significant advantage that expression vectors could be designed to receive the RDL-derived gene without the introduction of any extraneous residues into the coding sequence.12,26 Synthesis of ELP Genes by RDL. We used RDL to produce three ELP gene libraries. Within each library, genes encode the same repeat unit, as defined by the monomer gene (Figure 2), but differ in the number of repeats. The first library, ELP[V5], is comprised of a set of Val-Pro-Gly-ValGly homopolymers ranging in length up to 120 pentapeptides (∼50 kDa). The second library, ELP[V5A2G3], is a more complex copolymer that contains Val, Ala, and Gly in a 5:2:3 ratio at the fourth residue of the ELP pentapeptide repeat. This library encodes polypeptides ranging up to 330 pentapeptides (∼130 kDa) in length. The third library, ELP[V1A8G7], is a set of copolymers with a Val:Ala:Gly guest residue ratio of 1:8:7, ranging up to 320 pentapeptides (∼120 kDa). For the two copolymers, we dispersed the different guest residues throughout each sequence in order to minimize repetition at the gene level and to produce a pseudorandom distribution in the expressed ELP. Preferred E. coli codons were favored wherever possible,27 with exceptions made to

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Figure 5. ELP library produced by RDL. (A) Agarose gel (1.2%) electrophoresis of ELP[V5A2G3] genes visualized by ethidium bromide staining. The left lane contains a size standard, which is labeled in bp. Plasmids containing the ELP genes were digested with EcoR I and HinD III, producing a vector fragment (2635 bp) and an ELP gene fragment. The expected size of each ELP gene is labeled in bp on the right. The number of pentapeptide repeats encoded by each gene is labeled below each lane. (B) SDS-PAGE (4-20% gradient) visualized by copper staining of purified ELPs expressed from the genes in (A). The left lane contains a molecular weight standard, which is labeled in kilodaltons. The expected molecular weight of each ELP is indicated on the right. The length in pentapeptides of each ELP is labeled below each lane.

reduce repetition of the nucleotide sequence. We chose different monomer gene lengths for each ELP sequence, depending on the desired incremental step size during oligomerization by RDL. For simplicity, we discuss the synthesis of ELP[V5A2G3] only, although similar results were obtained for the other two gene libraries. Figure 5A shows the results of DNA agarose gel electrophoresis of the genes for the entire ELP[V5A2G3] library, which was synthesized as follows. The monomer gene cassette, which encodes 10 pentapeptides, was constructed by annealing complementary, chemically synthesized oligonucleotides and then ligated into pUC19 to yield pUC19ELP[V5A2G3-10]. In the first round of RDL, the monomeric ELP insert was prepared by digesting the pUC19-ELP[V5A2G3-10] vector with PflM I and Bgl I and purifying the insert after agarose gel electrophoresis. An ELP vector fragment was prepared to receive the insert by linearizing another aliquot of the same pUC19-ELP[V5A2G3-10] plasmid with only PflM I. The insert encoding the monomer gene was then ligated into the vector. Transformants yielded both pUC19-ELP[V5A2G3-20], the anticipated product, and also


pUC19-ELP[V5A2G3-30]. The latter construct was the product of a trimolecular ligation, created when two inserts were ligated into the linearized vector. Multiple inserts obtained in a single round of RDL are identical to the product obtained after sequential steps of RDL with single inserts, and for both cases, the ligation is seamless and the inserts are joined in a head-to-tail orientation. We have observed that for small inserts less than 500 bp, double inserts are commonly obtained for a small but significant fraction of transformants. This is useful because it reduces the number of RDL cycles required to build a larger library. After the initial round of RDL, the ELP[V5A2G3] library was expanded by recursive combination of the newly synthesized genes to produce larger genes. In the second round of RDL, an insert encoding 30 pentapeptides was prepared from pUC19-ELP[V5A2G3-30] by digestion with PflM I and Bgl I and was then ligated into PflM I linearized pUC19-ELP[V5A2G3-30] vector to yield the gene for 60 pentapeptide repeats. The 30 pentapeptide gene insert from the second round of RDL was then ligated into the pUC19ELP[V5A2G3-60] vector to form a gene encoding 90 pentapeptides. Next, the genes encoding 120, 150, and 180 pentapeptides were produced in parallel by ligating inserts encoding 30, 60, and 90 pentapeptides into the pUC19-ELP[V5A2G3-90] vector, respectively. Finally, genes encoding 240 and 330 pentapeptides were created by ligating the genes for ELP[V5A2G3-60] and ELP[V5A2G3-150] as inserts into the pUC19-ELP[V5A2G3-180] vector. Each RDL step requires minimal screening of transformants, and only 5 to 10 colonies are screened for each round. The percentage of positive clones in each RDL step ranges from ∼30 to 80% and appears to be independent of insert size. Additionally, ∼10 to 20% of screened colonies yield double inserts for inserts of ∼500 bp or less, although we have not observed double insertions for ELP genes larger than 500 bp. Therefore, when combining two genes of unequal size by RDL, it is desirable to use the smaller gene as the insert because of the possibility of creating tandem repeats in one round of RDL. To achieve these overall ligation efficiencies of up to 80%, however, it is critical to minimize WT background by ensuring that both digestion of the vector with RE1 (e.g., PflM I for the ELP genes) and its subsequent dephosphorylation are complete. The WT background could be eliminated completely using a third RE that cleaves the vector between its antibiotic resistance gene and the plasmid’s origin of replication, as described by Rosenfeld and Kelly.28 In this case, one fragment is prepared by digestion with RE1 and the third RE, and the second fragment is prepared by digestion with RE2 and the third RE. After purification of the two fragments and subsequent ligation, only the desired product produces a viable plasmid. This approach, however, would eliminate the possibility of obtaining multiple inserts in a single round of RDL. Expression of the ELP Gene Libraries. To express an oligomeric gene constructed by RDL, it is first excised from the cloning vector and ligated into an expression vector. The expression vector is designed to include a single, unique RE recognition site that provides RE1- and RE2-compatible cohesive ends upon cleavage. This site must allow ligation


of the RDL gene insert in frame with the reading frame of the expression vector. The expression vector also encodes other amino acids adjacent to the insertion site of the repetitive polypeptide gene. Minimal leader and trailer sequences might encode only the initiation and termination codons, respectively, while others can include codons for specific amino acids such as Lys or Cys to enable sitespecific chemical conjugation with the expressed polypeptide. Even more complex leading or trailing sequences can encode entire proteins, thereby producing fusion proteins.17,29 Transfer of the gene from the cloning vector to the expression vector is achieved using methods that are identical to gene oligomerization by RDL: an insert encoding the gene of interest, which is prepared by digestion of the cloning vector with RE1 and RE2, is ligated into the expression vector that has been linearized by single digestion with the insertion RE, which cleaves to produce cohesive ends that are compatible with those produced by RE1 and RE2. We have selected Sfi I as the insertion RE for all of our ELP expression vectors. The pET25b(+) T7 lac expression vector was therefore modified by replacement of the region between Nde I and EcoR I in the parent vector by oligonucleotide cassette mutagenesis to introduce a unique Sfi I site (Figure 2B). The Sfi I site was designed to be compatible with the RDL inserts, produced by digestion of the pUC19based cloning vectors with PflM I and Bgl I, by selecting the single stranded overhang produced by cleavage with Sfi I to be 5′-GGC-3′. When read in-frame with the ELP inserts, the Sfi I recognition sequence was designed to encode GlyPro immediately prior to the insertion site and Trp-Pro immediately following it. The Trp residue was chosen to allow measurements of the ELP concentration by UVvisible spectrophotometry. The remaining three residues were selected because they match well with the pentapeptide repeat of ELPs and are therefore unlikely to significantly affect the inverse temperature transition. We have constructed a number of different expression vectors, which can be divided into two categories: vectors for expression of “free” ELPs and vectors that enable expression of ELPs fused to other proteins.17,29 The vectors designed for expression of free ELPs typically encode two or three additional residues at the N- and C-termini of the ELP (Figure 2B). These residues are useful because they enable unique reactive side chains to be incorporated into the polypeptide for site-specific chemical conjugation, crosslinking, or surface immobilization of the ELP. These residues include Lys for conjugation with amine-reactive agents, Cys for sulfhydryl reactive agents, and N-terminal Ser or Thr (after in vivo processing of the initiating Met30 for reaction with hydrazide linkers after periodate oxidation.31 These expression vectors are generic in that they can be used to express any ELP sequence previously produced by RDL. Characterization of ELPs. Figure 5B shows SDS-PAGE results for the ELP[V5A2G3] library. When compared to a commercial MW marker, the ELPs consistently migrate as ∼20% larger than expected, a trend that has been previously observed by McPherson et al.10 However, the migration of the ELPs was consistent with the expected MW differences with respect to each other. Similar SDS-PAGE results were

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Figure 6. ELP Tt as a function of sequence and chain length. The Tt was determined by temperature-dependent turbidity measurements for each member of three ELP libraries: ELP[V5] (0), ELP[V5A2G3] (O), and ELP[V1A8G7] (4). The ELP concentration was 25 µM in PBS. The Tt was defined as the solution temperature at the maximum of the turbidity gradient obtained while heating the solution at a rate of 1 °C min-1. These data show that the exquisite control over both composition and chain length provided by RDL is critical for the design of ELPs with precisely specified thermal properties.

obtained for the other two libraries. To further verify the MWs of the ELPs, the following members of each ELP library were characterized by MALDI-MS: ELP[V5-120], ELP[V5A2G3-180], and ELP[V1A8G7-160]. The MW of each polypeptide as determined by MALDI-MS was within 0.4% of the calculated value. The thermal behavior of each member of the three ELP libraries was studied by measuring solution turbidity as a function of temperature. Upon heating the solution to the Tt, the solutions rapidly become cloudy due to ELP aggregation. We defined the Tt as the temperature at which the increase in turbidity was most rapid. Figure 6 shows the Tt values for each library as a function of ELP size. ELPs smaller than those shown (i.e., ELP[V5-15], ELP[V5A2G330], and ELP[V1A8G7-96]) were also studied by temperaturedependent turbidimetry but did not exhibit a transition below 90 °C, the highest temperature that was experimentally accessible. These constructs, however, do exhibit a thermal transition at lower temperatures when NaCl is added to the solution to depress their Tt values, showing that these ELPs with high Tt values were thermally responsive (data not shown). The data in Figure 6 show that the Tt is a function of two macromolecular parameters, both of which can be precisely controlled at the gene level through RDL. First, the Tt increases with decreasing ELP MW. This result qualitatively parallels that of Urry et al., who studied the inverse temperature transition of chemically synthesized ELPs at high concentration (∼40 mg/mL).22 However, the logarithmic relationship between Tt and MW that they observed is not reproduced for the ELPs in this study, which were studied at the significantly lower concentration of 25 µM (∼0.25 to 3.25 mg/mL). Second, for a given MW, decreasing the hydrophobicity of the guest residues increases the Tt, which is also consistent with previous results of Urry et al.21 On the basis of these data, it is now possible to design an ELP of specified sequence and MW to exhibit a desired Tt. Because the thermal properties of ELPs depend on their sequence and chain length, the synthesis of ELPs by RDL

Meyer and Chilkoti

provides an excellent example of the utility of this synthesis methodology. ELP Block Copolymer. The simple and facile synthesis of block copolymers is another example of the utility of RDL, which provides exquisite control both over the architecture of the block copolymer and over the repeat sequence within individual blocks. To demonstrate this application of RDL, we synthesized and expressed a gene encoding an AB diblock copolymer, in which ELP[V1A8G7-64] is followed seamlessly by ELP[V5-60]. This was achieved in one cycle of RDL using the PflM I/Bgl I-digested ELP[V5-60] gene as the insert and pUC19-ELP[V1A8G7-64] as the PflM I-linearized vector. Both of these genes had been previously generated during synthesis of the ELP libraries. We chose these two blocks because ELP[V5-60] has a Tt of 35 °C and ELP[V1A8G7-64] has a Tt > 90 °C, and we hypothesized that a copolymer of these two blocks would form a nanoparticle in solution as a function of temperature, driven by the disparity in the Tt of each block. If the two blocks exhibited independent transition behavior, the ELP[V5-60] block should hydrophobically collapse and aggregate at temperatures above its Tt while the ELP[V1A8G7-64] block should remain hydrophilic and solvated, leading to the formation of a micellar structure.32,33 We selected ELP[V5A2G3-120], a pseudorandom analogue, as a control sequence for comparison to the ELP[V1A8G764]-ELP[V5-60] block copolymer. The block copolymer is 124 pentapeptides in length and its guest residues are composed of 51.6% Val, 25.8% Ala, and 22.6% Gly, with the N-terminal block primarily composed of Ala and Gly guest residues followed by a block containing exclusively Val guest residues. Similarly, the pseudorandom ELP[V5A2G3-120] is 120 pentapeptides in length and its guest residues are composed of 50% Val, 20% Ala, and 30% Gly. In contrast to the block copolymer, however, the guest residues are dispersed evenly throughout the polymer chain. Thus, comparison of the block and pseudorandom copolymer was expected to provide insight into the effect of the distribution of guest residues within an ELP sequence on its thermal behavior. The block and pseudorandom copolymers were each studied by measuring solution turbidity and by DLS as a function of temperature (Figures 7 and 8). When a solution of the pseudorandom ELP[V5A2G3-120] copolymer is heated, reaching the Tt of 44.6 °C triggers a stepwise increase in turbidity (Figure 7). DLS indicates that this increase in turbidity results from the conversion of soluble ELP monomer with a hydrodynamic radius (Rh) of 4.8 ( 1.1 nm (mean Rh ( polydispersity) to aggregates with a Rh of 1.2 ( 0.26 µm (Figure 8A). We term this transition from soluble monomer to micrometer-size aggregates the “bulk transition” because it results in the sudden and dramatic formation of aggregates over a narrow temperature range without formation of particles of intermediate size. A solution of the ELP[V1A8G7-64]-ELP[V5-60] block copolymer displays a bulk transition at 50.8 °C, also leading to the formation of micrometer size aggregates. The disparity between the bulk Tt values of the pseudorandom copolymer (Tt ) 44.6 °C) and the block copolymer suggests that the


Figure 7. Solution turbidity of ELP block and pseudorandom copolymers as a function of temperature. OD350 as a function of temperature for solutions of three ELPs: ELP[V5-60], ELP[V5A2G3120], and the ELP[V1A8G7-64]-[V5-60] block copolymer. The turbidity profiles were obtained for 25 µM ELP concentration in PBS, while heating at a rate of 1 °C min-1 (solid lines). All solutions cleared fully upon cooling; however, for clarity, a cooling profile is shown only for ELP[V1A8G7-64]-[V5-60] (dashed line). The ELP[V1A8G7-64]-[V5-60] block copolymer exhibited a complex turbidity profile, with three inflection points observed over a range of 10 °C.

Figure 8. Particle size as a function of temperature for the (A) pseudorandom and (B) block ELP copolymers. The hydrodynamic radii of each ELP was measured by dynamic light scattering as a function of temperature (mean ( polydispersity of the particle size distribution). The corresponding turbidity profiles from Figure 7 are replotted for comparison with the DLS results (solid lines). These results suggest that the block copolymer forms a micelle-like structure at temperatures intermediate between the initial collapse of the more hydrophobic ELP segment at 40.0 °C and the collapse and subsequent aggregation of the less hydrophobic segment at 50.8 °C.

bulk aggregation behavior of an ELP copolymer is sensitive to the distribution of the guest residues in the polymer chain. The bulk Tt at 50.8 °C of the block copolymer is also significantly different from that of each free block. The bulk transition of the block copolymer occurs well below the Tt


of ELP[V1A8G7-64], which is >90 °C, indicating that fusion to the more hydrophobic ELP[V5-60] reduces the Tt of the ELP[V1A8G7-64] segment. Conversely, the first detectable change in turbidity of the block copolymer (∼40 °C, as discussed below) is above the bulk transition at 34.8 °C of the ELP[V5-60] homopolymer, which comprises the hydrophobic, low Tt segment of the block copolymer. This shows that fusion of ELP[V5-60] to the more hydrophilic ELP[V1A8G7-64] sequence in the block copolymer significantly increases the Tt of the ELP[V5-60] segment. In contrast to the random copolymer, which simply displays a single transition from monomer to micrometer aggregates, the ELP block copolymer exhibits temperaturedependent mesoscale self-assembly. In addition to the bulk transition at 50.8 °C, the turbidity profile of the block copolymer also exhibits two inflection points at lower temperatures that are not observed for the ELP[V5-60] homopolymer or the pseudorandom ELP[V5A2G3-120] copolymer. The first inflection point in the turbidity profile of the block copolymer is observed at 40.0 °C, and between 40 and 47.5 °C, a linear increase in turbidity is observed, with a slope that is significantly different from baseline. The DLS results for ELP[V1A8G7-64]-ELP[V5-60] show that the ELP monomer (4.4 ( 1.6 nm) is the sole species in solution at temperatures below 40 °C (Figure 8B). As the temperature is increased, a new, larger particle with a Rh of 20.4 ( 5.8 nm is observed at 40 °C that persists up to 47 °C. At 47.5 °C, a second inflection point in the turbidity versus temperature profile of the block copolymer is observed, and the DLS data show a discontinuous jump in the Rh of the particles from 20.4 ( 5.8 nm to 54.5 ( 20.3 nm. These particles are stable until the bulk transition occurs at 50.8 °C, above which larger aggregates with a Rh of 1.4 ( 0.35 µm are formed. When the solution is cooled, the turbidity profile closely overlays the heating curve, showing that all three inflections at 40.0, 47.5, and 50.8 °C are due to fully reversible transitions. Note that the presence of smaller particles can be masked in the DLS data by scattering from larger particles, even though several species may coexist at a given temperature. The turbidity and DLS results for the ELP[V1A8G7-64]ELP[V5-60] copolymer suggest that the two blocks undergo sequential and independent transitions at different temperatures. We hypothesize that upon increasing the temperature to 40.0 °C, the solvated ELP[V5-60] block undergoes an inverse temperature transition, resulting in desolvation and hydrophobic collapse of this segment of the polymer chain. Driven by hydrophobic interactions between the collapsed ELP[V5-60] segments, molecules of the block copolymer then self-assemble to form nanoparticles with a Rh of 20 nm that are composed of a hydrophobic core of collapsed ELP[V5-60] surrounded by a hydrophilic shell of solvated ELP[V1A8G7-64] segments. The initial appearance of these micelle-like nanoparticles causes a slight increase in turbidity. The linear increase in solution turbidity observed as the temperature is increased between 40.0 and 47.5 °C is likely caused by an increase in the concentration of nanoparticles at the expense of monomeric ELP molecules. These nanoparticles, which have a constant diameter of ∼40 nm over

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this temperature range, undergo a rearrangement at 47.5 °C to form nanoparticles that have an apparent diameter of ∼110 nm. The mechanism of the transition leading to an increase in the diameter of the nanoparticles from ∼40 to ∼110 nm is not known at this time. Finally, at 50.8 °C, the solventexposed ELP[V1A8G7-64] block undergoes its inverse temperature transition, which drives the aggregation of the ∼110 nm diameter nanoparticles to form micrometer-sized aggregates. Advantages of RDL. RDL is a facile method to rapidly generate a library of polymers with systematically varied MWs. This approach is flexible in that the insert and vector for a given round of RDL can be prepared from the same plasmid or from different plasmids. When the insert and linearized vector are prepared from the same plasmid, each round of RDL doubles the size of the gene. Repeated dimerization of a gene is the most rapid method to generate a large, repetitive gene. Each round of RDL can be completed in 2 days, and gene libraries encoding large polypeptides can be generated in just a few weeks. For example, a 6400 bp gene composed of 64 repeats of a 100 bp monomer can be constructed in six sequential rounds of RDL. In practice, large genes can be produced in even fewer rounds because multiple inserts are often obtained in the earlier rounds of RDL. RDL is not, however, restricted solely to the dimerization of a gene. It is a modular and flexible synthesis technique that allows any two gene sequences to be seamlessly combined in a defined orientation, requiring only that each sequence is designed to incorporate compatible RE recognition sites at their 5′ and 3′ termini. Ligation of the two gene sequences results in a longer sequence that can itself be used in subsequent rounds of RDL. For example, instead of dimerization, DNA oligomers of different lengths can be joined in a round of RDL (e.g., a trimer with a tetramer to yield a heptamer), which enables the construction of a gene of any length in increments of the monomer gene size. Because RDL is a modular synthesis methodology, two genes encoding different sequences can also be combined to form a larger, more complex sequence that can then be oligomerized in subsequent rounds of RDL (e.g., as illustrated by the right pathway in Figure 1). Alternatively, larger genes with different sequences previously constructed by RDL can be combined to produce block copolymers, as described for ELP[V1A8G7-64]-ELP[V5-60]. Compared to previously reported methods for the assembly of synthetic genes encoding repetitive polypeptides, RDL has a number of unique features that account for its flexibility and precision. First, RDL enables a desired MW to be rationally and precisely targeted during synthesis. Second, each round of RDL yields identical DNA oligomers that do not have to be fractionated or screened (with the exception of a low fraction of multimers in early rounds of RDL with shorter gene segments, as described above). Third, the process of assembling a large gene by RDL necessarily creates a library of potentially useful smaller genes ranging in size from the monomer to the target gene. Finally, the procedure is modular in that monomers or oligomers encod-

Meyer and Chilkoti

ing different peptide or protein repeats can be combined at any step to further generate diversity at the sequence level. RDL is also useful in the synthesis of very large repetitive genes that are several thousand nucleotides in length or greater. We have constructed genes up to 4950 bp in length (33 repeats of a 150 bp monomer gene), encoding an ELP with a MW of ∼130 kDa. Although this is the largest gene we have attempted to synthesize to date, this does not represent an upper limit. This is because, regardless of the gene size, an RDL step requires the ligation of only two DNA fragments. In contrast, producing large genes by a concatamerization method is complicated by the likelihood of circularization of larger oligomers. The resulting closedcircle oligomer cannot be subsequently ligated into a vector. Methods to ameliorate this problem have been proposed, including the use of chain-terminating capping sequences,10 sequential concatamerization,25 and ligation under fluid shear;34 however reports of the successful synthesis of very large, repetitive genes by concatemerization are nonetheless limited. The modular nature of RDL provides a convenient and powerful method to vary the physicochemical properties of block copolymers, which are of great interest for drug and gene delivery.35,36 For example, RDL should prove useful in precisely engineering the properties of ELP block copolymers to enable thermally triggered nanoparticle formation and to control the nanoparticle size and its drug loading capacity. These parameters can be systematically varied by selecting ELP blocks of different lengths and sequences, and hence Tt values. The loading of drugs could be maximized by optimizing noncovalent interactions between the hydrophobic segment of the block copolymer and the drug. Sites for chemical conjugation of the drug could also be engineered into either the low or high Tt block. Similarly, DNA for gene delivery could be bound within the ELP nanoparticle by polycationic blocks. Altering the particle size by adjusting the relative lengths of each ELP block will enable control of the pharmacokinetics of systemically injected, drug-loaded nanoparticles. RDL should also enable the presentation of affinity targeting peptides or proteins at the termini of the solvent-exposed hydrophilic segments to enable receptormediated targeting of the nanoparticle to physiological targets of interest. Furthermore, mixing of block copolymers containing a targeting peptide (or peptides) with polymers lacking a targeting sequence would enable the number of targeting moieties per nanoparticle to be precisely specified and would thereby enable polyvalency effects to be exploited in targeted drug delivery.37 More broadly, RDL is a generic synthesis approach that is not restricted to ELPs and can be used to oligomerize many other genes of interest. For example, RDL can be used to systematically vary the location and density of cell attachment sequences or cross-linking sites in repetitive polypeptides designed as tissue engineering scaffolds. Similarly, oligomers of peptide pharmaceuticals can be produced by RDL in order to increase the avidity of drugs for their targets. RDL is also useful for applications in which DNA oligomers are the end product, such as the synthesis of multiple copies of a therapeutic gene for gene delivery or antisense therapy.



In conclusion, RDL is a useful strategy to generate oligomeric genes and polypeptides for diverse applications in medicine and biotechnology. Acknowledgment. This work was supported by grants from the Whitaker Foundation and the National Institutes of Health (R21-GM-057373 and R01-GM-61232). We also thank the Whitaker Foundation for support of D.E.M. as a graduate fellow.

(17) (18) (19) (20) (21) (22) (23)

References and Notes (1) Cappello, J. Trends Biotechnol. 1990, 8, 309-311. (2) McGrath, K. P.; Tirrell, D. A.; Kawai, M.; Mason, T. L.; Fournier, M. J. Biotechnol. Prog. 1990, 6, 188-192. (3) Barron, A. E.; Zuckermann, R. N. Curr. Opin. Chem. Biol. 1999, 3, 681-687. (4) Nagarsekar, A.; Ghandehari, H. J. Drug Target. 1999, 7, 11-32. (5) McPherson, D. T.; Morrow, C.; Minehan, D. S.; Wu, J.; Hunter, E.; Urry, D. W. Biotechnol. Prog. 1992, 8, 347-352. (6) Feeney, K. A.; Tatham, A. S.; Gilbert, S. M.; Fido, R. J.; Halford, N. G.; Shewry, P. R. Biochim. Biophys. Acta 2001, 1546, 346-355. (7) Kostal, J.; Mulchandani, A.; Chen, W. Macromolecules 2001, 34, 2257-2261. (8) Cappello, J.; Crissman, J.; Dorman, M.; Mikolajczak, M.; Textor, G.; Marquet, M.; Ferrari, F. Biotechnol. Prog. 1990, 6, 198-202. (9) Creel, H. S.; Fournier, M. J.; Mason, T. L.; Tirrell, D. A. Macromolecules 1991, 24, 1213-1214. (10) McPherson, D. T.; Xu, J.; Urry, D. W. Protein Expr. Purif. 1996, 7, 51-57. (11) Fukushima, Y. Biopolymers 1998, 45, 269-279. (12) McMillan, R. A.; Lee, T. A. T.; Conticello, V. P. Macromolecules 1999, 32, 3643-3648. (13) Urry, D. W. Prog. Biophys. Mol. Biol. 1992, 57, 23-57. (14) Urry, D. W. J. Phys. Chem. B 1997, 101, 11007-11028. (15) Cappello, J.; Crissman, J. W.; Crissman, M.; Ferrari, F. A.; Textor, G.; Wallis, O.; Whitledge, J. R.; Zhou, X.; Burman, D.; Aukerman, L.; Stedronsky, E. R. J. Controlled Release 1998, 53, 105-117. (16) Urry, D. W.; Pattanaik, A.; Xu, J.; Woods, T. C.; McPherson, D. T.;

(24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) (36) (37)

Parker, T. M. J. Biomater. Sci. Polym. Ed. 1998, 9, 1015-1048. Meyer, D. E.; Chilkoti, A. Nat. Biotechnol. 1999, 17, 1112-1115. Urry, D. W. Trends Biotechnol. 1999, 17, 249-257. Welsh, E. R.; Tirrell, D. A. Biomacromolecules 2000, 1, 23-30. Meyer, D. E.; Kong, G. A.; Dewhirst, M. W.; Zalutsky, M. R.; Chilkoti, A. Cancer Res. 2001, 61, 1548-1554. Urry, D. W.; Luan, C.-H.; Parker, T. M.; Gowda, D. C.; Prasad, K. U.; Reid, M. C.; Safavy, A. J. Am. Chem. Soc. 1991, 113, 43464348. Urry, D. W.; Trapane, T. L.; Prasad, K. U. Biopolymers 1985, 24, 2345-2356. Ausubel, F. M.; Brent, R.; Kingston, R. E.; Moore, D. H.; Seidman, J. G.; Smith, J. A.; Struhl, K. Current Protocols in Molecular Biology; John Wiley: New York, 1995. Lee, C.; Levin, A.; Branton, D. Anal. Biochem. 1987, 166, 308312. Lee, J. H.; Skowron, P. M.; Rutkowska, S., M.; Hone, S. S.; Kim, S. C. Genet. Anal. 1996. Padgett, K. A.; Sorge, J. A. Gene 1996, 168, 31-35. Aota, S.-i.; Gojobori, T.; Ishibashi, F.; Maruyama, T.; Ikemura, T. Nucleic Acids Res. 1988, 16, Suppl., r315-402. Rosenfeld, P. J.; Kelly, T. J. J. Biol. Chem. 1986, 261, 1398-1408. Meyer, D. E.; Trabbic-Carlson, K.; Chilkoti, A. Biotechnol. Prog. 2001, 17, 720-728. Hirel, P. H.; Schmitter, J. M.; Dessen, P.; Fayat, G.; Blanquets, S. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 8247-8251. Geoghegan, K. F.; Stroh, J. G. Bioconjugugate Chem. 1992, 3, 138146. Chung, J. E.; Yokoyama, M.; Aoyagi, T.; Sakurai, Y.; Okano, T. J. Controlled Release 1998, 53, 119-130. Lee, T. A. T.; Cooper, A.; Apkarian, R. P.; Conticello, V. P. AdV. Mater. 2000, 12, 1105-1110. Haber, C.; Wirtz, D. Biophys. J. 2000, 79, 1530-1536. Kataoka, K.; Harada, A.; Nagasaki, Y. AdV. Drug DeliV. ReV. 2001, 47, 113-131. Jones, M.; Leroux, J. Eur. J. Pharm. Biopharm. 1999, 48, 101111. Mammen, M.; Choi, S.-K.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 2454-2794.

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Genetically Encoded Synthesis of Protein-Based Polymers with

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