Improved Assembly of Multimeric Genes for the Biosynthetic

May 29, 2002 - Biomacromolecules , 2002, 3 (4), pp 874–879. DOI: 10.1021/bm0255342 .... Introducing a combinatorial DNA-toolbox platform constitutin...
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Biomacromolecules 2002, 3, 874-879

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Improved Assembly of Multimeric Genes for the Biosynthetic Production of Protein Polymers Nichole L. Goeden-Wood,† Vincent P. Conticello,‡ Susan J. Muller,† and Jay D. Keasling*,† Department of Chemical Engineering, University of California at Berkeley, Berkeley, California 94720-1462; and Department of Chemistry, Emory University, Atlanta, Georgia 30322 Received March 6, 2002; Revised Manuscript Received April 18, 2002

We report a general method for the construction of highly repetitive synthetic genes and their use in the biosynthetic production of artificial protein polymers. Through the application of improved recombinant DNA techniques and high-throughput screening methods, we have developed a facile approach to rapid gene assembly and cloning which is widely applicable in the biosynthesis of novel protein polymers. Using this technique, synthetic genes encoding tandem repeats of the β-sheet forming amino acid sequence AEAEAKAK were constructed and subsequently cloned into a bacterial expression host for inducible protein production. A 17-kDa fusion protein, poly-EAK9, was isolated from Escherichia coli and purified to homogeneity by immobilized metal affinity chromatography. The amino acid sequence and molecular weight were confirmed by amino acid analysis, N-terminal sequencing, and MALDI-TOF mass spectrometry. Circular dichroism studies on the artificial protein poly-EAK9 demonstrate the formation of a β-sheet structure in aqueous solution. Introduction Genetic and protein engineering have emerged as powerful tools for the construction of macromolecules.1-4 With the development of recombinant DNA techniques, it is now possible to construct genes encoding protein polymers composed of naturally occurring or even nonnatural amino acid sequences.5-19 Although recent progress has been made in living polymerization techniques,20 the protein synthetic machinery of the cell remains unsurpassed in its ability to precisely control polymer chain length, composition, sequence, and stereochemistry. The ability to control these properties provides unique opportunities for the polymer scientist to design new materials and explore their structureproperty relationships. Polydispersity is no longer a factor, variations in primary sequence are eliminated, and functional groups can be placed at precise locations along the chain. However, to date, a relatively small number of high molecular weight artificial protein polymers have been synthesized by bacterial expression. The biosynthesis of novel protein polymers can be a lengthy and time-consuming process, as it typically requires the construction and cloning of large synthetic genes encoding tandem repeats of target amino acid sequences.16,21 In our work, a concerted effort has been made to improve the speed and efficacy of the cloning process through the application of improved recombinant DNA techniques and high-throughput screening methods. The rapid construction of synthetic genes will facilitate the development and screening of large numbers * To whom all correspondence should be addressed. E-mail: keasling@ socrates.berkeley.edu. † University of California at Berkeley. ‡ Emory University.

of novel protein polymers for use in advanced material applications. A general method for the assembly of synthetic genes based on the Seamless cloning technique was reported previously.21 In this method, double-stranded oligonucleotide cassettes (DNA “monomers”) are enzymatically oligomerized to form large synthetic genes (DNA “multimers”) encoding tandem repeats of target amino acid sequences. The Type IIs restriction endonuclease, Eam1104 I, is utilized for direct cloning of the DNA multimer into the recipient expression vector. Unlike earlier methods, this technique can be used to directionally clone any target DNA sequence without relying on a limited pool of nonpalindromic restriction enzymes to achieve head-to-tail ligation of the DNA monomers. Furthermore, since the Eam1104 I recognition sites are eliminated from the target DNA upon cleavage, only the DNA encoding the desired amino acid sequence is joined in the subsequent ligation reaction. Although the use of Eam1104 I results in fewer subcloning steps and avoids the incorporation of extraneous nucleotides, internal Eam1104 I recognition sites are commonly found in expression vectors such as pET-19b (Novagen). To prevent undesired cleavage of the plasmid DNA, 5′-methyldeoxycytosine (5mdCTP) has previously been used in the PCR amplification of the expression vector. The methylated dCTP is incorporated into the plasmid’s internal Eam1104 I recognition sites and not the primer-derived Eam1104 I sites, preventing internal cleavage of the plasmid DNA upon treatment with Eam1104 I. It has been shown, however, that the incorporation of 5mdCTP is incomplete, resulting in subsequent fragmentation of the expression vector as reported previously.21

10.1021/bm0255342 CCC: $22.00 © 2002 American Chemical Society Published on Web 05/29/2002

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Improved Assembly of Multimeric Genes

Figure 1. Design of DNA monomer encoding the amino acid sequence [AEAEAKAK]2.

To alleviate this problem, a new expression vector was constructed by site directed mutagenesis in order to remove the internal cleavage sites and eliminate the need for 5′-methyldeoxycytosine. An alternative but equally effective Type IIs restriction endonuclease, Sap I, was selected for use due to the presence of a longer recognition sequence (7 bp instead of the 6 bp Eam1104 I recognition sequence), which reduced the probability of multiple recognition sites located within the target DNA. The plasmid, pET-19b, contains three Eam1104 I sites and only one Sap I site, making Sap I more amenable to removal by site directed mutagenesis. By eliminating the fragmentation of the recipient expression vector, pET-19b, our ability to effectively clone large DNA multimers into the plasmid DNA increased significantly. As a result, a large number of Escherichia coli clones were generated, requiring efficient screening to identify clones containing genes of the desired molecular weight. For this purpose, a rapid and high-throughput method was developed which utilized 96-well plates for all steps of the screening process. Synthetic genes present in less than 1% of the screened population were rapidly and easily identified. This method was used to construct a self-assembling amphiphilic polymer, poly-EAK9, based on the alternating amino acid repeat sequence AEAEAKAK. Protein sequences of alternating hydrophilic (e.g., Glu and Lys) and hydrophobic residues (e.g., Ala) are known to adopt β-sheet structures in aqueous solutions, and previous work on ionic self-complementary peptides has demonstrated their ability to self-assemble into macroscopic membranes upon the addition of monovalent metal ions.22 These synthetic peptide membranes, however, are relatively fragile and difficult to handle.23 Therefore, through the use of recombinant DNA techniques, we have constructed a high molecular weight analogue of the EAK sequence to probe its self-assembly and mechanical properties for potential use as a biomaterial. Materials Synthetic oligonucleotides were obtained from Operon, Inc. (Alameda, CA). E. coli strains DH10B and TOPO10F′ were purchased from Invitrogen, Inc. (Carlsbad, CA). The plasmid, pBluescript SK II, PFU Turbo polymerase, and the QuikChange site-directed mutagenesis kit were obtained from Stratagene, Inc. (La Jolla, CA). Pellet Paint, pET-19b, and the E. coli strain used, BL21(DE3), were obtained from Novagen, Inc. (Madison, WI). Pre-cast acrylamide gels in 1X TBE buffer were purchased from Bio-Rad Laboratories (Hercules, CA). The Plasmid MiniPrep kit and the Qiaquick PCR purification kit were obtained from Qiagen, Inc. (Valencia, CA). The 96-well Multiscreen FB and NA plates

and the vacuum manifold were obtained from Millipore, Inc (Bedford, MA). Dideoxy sequencing was performed by Davis Sequencing, Inc. (Davis, CA). DNA concentrations were measured using a Beckman DU 640 spectrophotometer. All other reagent enzymes were purchased from New England Biolabs (Beverly, MA). Protein purification was performed using the AKTA FPLC (Amersham Pharmacia) with nickel-chelated sepharose. Amino acid compositional analysis, protein sequencing, and MALDI-TOF mass spectrometry were performed by the UC Davis Molecular Structure Facility (Davis, CA). Circular dichroism spectra were recorded using an AVIV 62DS spectrometer and a 1.0 mm quartz cuvette. Methods DNA Monomer Construction. A DNA sequence was designed to encode the “EAK” repeat unit [AEAEAKAK]2 and to include flanking restriction sites for efficient cloning (Figure 1). The monomeric DNA was assembled from chemically synthesized oligonucleotides 5-GCTGAAGCTGAGGCTAAGGCCAAGGCAGAAGCTGAAGCGAAGGCTAAAGCT-3′ and 5′-GCTTTAGCCTTCGCTTCAGCTTCTGCCTTGGCCTTAGCCTCAGCTTCAGC-3′. The oligos (10 µg each) were heated using a PCR thermocycler (PerkinElmer) in 1X BamH I buffer (200 µL) at 95 °C for 7 min and then slowly cooled to 4 °C at 0.1 °C/s. Annealed oligos were enzymatically digested during an overnight incubation with BamH I (100 units), EcoR I (100 units), and BSA (1 mg/mL) at 37 °C. Digested duplex DNA was isolated by preparative gel electrophoresis (2.5% Nusieve GTG agarose, 1X TBE buffer) and purified using the Mermaid spin filter kit (Q-BioGene). The recipient plasmid, pBluescript SK II (10 µg), was enzymatically digested during an overnight incubation in 1X BamH I buffer with BamH I (100 units), EcoR I (100 units), and BSA (1 mg/mL). Calf intestinal alkaline phosphatase (CIAP; 10 units) was added to the reaction mixture and allowed to incubate for an additional 4 h. Following the incubation period, the sample was heated to 75 °C for 10 min to inactivate the calf intestinal alkaline phosphatase. The digested plasmid DNA was purified using the Qiaquick spin filter kit (Qiagen, Inc.) and eluted in 30 µL of 10 mM TrisHCl (pH 8). Using T4 DNA ligase (400 units), the monomeric DNA (100 ng) and the plasmid DNA (20 ng) were enzymatically ligated at 16 °C for 16 h in 1X T4 DNA ligase buffer (10 µL) supplemented with 10 mM rATP. The ligation mixture (1 µL) was used to transform electrocompetent E. coli strain DH10B cells (20 µL). Electrotransformed cells were plated on LB agar plates containing ampicillin (100 µg/mL) and

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X-gal (32 µg/mL) and incubated for approximately 16 h. Positive transformants (white colonies) were cultured overnight in LB medium (5 mL) with ampicillin (100 µg/mL). Cells were harvested and the plasmid DNA was isolated using the Plasmid Miniprep kit (Qiagen, Inc.). Purified plasmid DNA (0.8 µg) was enzymatically digested with BamH I (5 units) and EcoR I (5 units) in 1X BamH I buffer (10 µL) supplemented with BSA (1 mg/mL) for 1 h at 37 °C. Digested plasmid DNA was examined by gel electrophoresis (TBE pre-cast agarose gel, 1X TBE buffer), and the presence of the DNA monomer was identified by restriction fragment analysis. Using the T3 and T7 universal primers, dideoxy sequencing was performed on a positive clone, pBlue-EAK, to confirm the sequence of the DNA monomer prior to gene assembly. Preparation and Polymerization of DNA Monomers. Amplification of the monomer DNA was achieved by growth of E. coli DH10B cells, containing the pBlue-EAK plasmid, in LB medium (500 mL) with ampicillin (100 µg/mL) for 12-16 h. Plasmid DNA was purified using the Qiagen Maxiprep procedure (Qiagen, Inc.). The purified plasmid DNA (250 µg) was incubated 12 h with the restriction endonuclease Eam1104 I (2.5 units/µg DNA) in 1X Eam1104 I buffer (1.25 mL). The DNA was extracted with phenol/ chloroform (1:1) and was ethanol precipitated using the coprecipitant Pellet Paint (2 µL). The DNA was resuspended in 10 mM Tris-HCl (pH 8) to a final concentration of 0.5-1 mg/mL. Isolation of the monomer DNA fragment was achieved by preparative gel electrophoresis (2.5% Nusieve GTG agarose, 1X TBE buffer). The desired band was excised from the gel and incubated in an equal volume of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8) for 30 min at room temperature. The monomer DNA was separated from the agarose gel by passage through an Ultrafree CL centrifuge filter (Millipore, 0.45 µm) at 5000g for 5 min. The filtrate was ethanol precipitated using Pellet Paint, and the monomer DNA was resuspended in 10 mM Tris-HCl, pH 8, to a final concentration of approximately 25 ng/µL. Self-ligation of purified monomer DNA (2 µg) was achieved by an overnight incubation with highly concentrated T4 DNA ligase (2000 units) in 1X T4 DNA ligase buffer (80 µL) supplemented with 10 mM rATP at 16 °C. DNA multimers were separated by preparative gel electrophoresis (1% Seaplaque agarose, 1X TAE) and multimers of the desired molecular weight were excised from the gel, purified using the Zymoclean gel DNA recovery kit (Zymos Research), and eluted with two 8 µL aliquots of sterile water. The concentration of the eluted sample was estimated using a DNA dipstick kit (Invitrogen, Inc.) designed for measurement of dilute DNA solutions. Purified DNA multimers were then cloned into the recipient expression vector, pET-19b (Novagen). Construction of the Recipient Expression Vector. QuikChange site-directed mutagenesis (Stratogene) was performed on the plasmid, pET-19b, to eliminate the extraneous Sap I site. The mutagenized pET-19b plasmid was further amplified by inverse PCR28 to generate compatible termini to the DNA multimers. Five PCR reactions (100 µL each) were prepared containing 1X PFU Turbo Buffer,

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plasmid DNA (10 ng), dNTPs (200 µM each), primers (100 ng each), and PFU Turbo polymerase (2.5 units). Samples were heated to 95 °C (45 s), and cycled (27×) at 95 °C (45 s), 60 °C (45 s), and 72 °C (10 min), with a final extension time at 72 °C (10 min). PCR samples were cooled to 4 °C, and the amplified DNA was isolated via preparative gel electrophoresis (1% Seaplaque agarose, 1X TAE buffer). A band corresponding to the desired molecular weight was excised from the gel and purified using the Qiaquick PCR Purification spin filters. Purified DNA (10 µg) was digested with Sap I (25 units) in 1X NEB Buffer 4 (200 µL) for 12 h. Following the incubation period, calf intestinal alkaline phosphatase, CIAP (5 units), was added to the reaction mixture, incubated an additional 4 h, and purified as described earlier for CIAP-treated plasmid DNA. Cloning and High-Throughput Screening of DNA Multimers. The Eam1104 I treated DNA multimers (∼100 ng) and the Sap I and CIAP-treated plasmid DNA (20 ng) were enzymatically ligated with T4 DNA ligase (400 units) in 1X T4 DNA ligase buffer (20 µL) supplemented with 10 mM rATP. The ligation reaction was incubated overnight (16 °C) and used to electrotransform competent cells of E. coli strain DH10B. Transformed cells (10-100 µL) were plated on LB agar plates containing ampicillin (100 µg/mL) and incubated for 16 h at 37 °C. Positive transformants were selected and used to inoculate 1 mL of 2xYT medium with ampicillin (100 µg/mL) in 96deep well plates (2 mL) containing a silica bead per well for improved agitation. Plates were incubated 20-24 h at 320 rpm. At harvest time, freezer stocks were prepared by transferring 70 µL of each culture to a separate 96-well plate (200 µL) containing 30 µL of 50% sterile glycerol per well. The remaining cells were harvested by centrifugation at 1000g for 10 min (Sorvall SH-3000 microplate rotor) and subjected to a standard alkaline lysis and RNase A treatment.24 Cell lysate (∼240 µL) was loaded onto the Multiscreen NA lysate clearing plate (Millipore), and the plasmid DNA was separated by vacuum filtration (8 in. Hg). Plasmid DNA filtrates were eluted into the Multiscreen FB binding plate (Millipore) containing 150 µL of 8 M Guanidine-HCl (binding solution). Samples were mixed by repeated pipetting, and vacuum was applied (20 in. Hg) to separate the binding solution from the plasmid DNA. Bound plasmid DNA was washed twice with 80% ethanol and the FB plate was allowed to dry completely under vacuum. DNA was resuspended in two 30 µL aliquots of TE buffer and eluted into a 96-well, conical bottom collection plate (200 µL) using vacuum filtration (20 in. Hg). Purified plasmid DNA (8 µL) was transferred to a 96well, V-bottom plate, containing 1 µL of 10X NEB Buffer 2, and the restriction endonucleases, Nde I (0.5 µL) and Hind III (0.5 µL). Samples were incubated for 2 h at 37 °C and then analyzed by gel electrophoresis using a 100-well, 1% agarose gel in 1X TAE buffer (Bio-Rad Sub-Cell model 192 Cell). Plasmid DNA containing high molecular weight inserts were identified by restriction fragment analysis and confirmed by dideoxy sequencing with both forward and reverse primers based on the T7 promoter and terminator sequences.

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Protein Expression. The plasmid pET19-EAK9 was used to transform chemically competent cells of E. coli strain BL21(DE3). Transformed cells were incubated in LB medium containing 10 mM MgCl2 for 1 h at 37 °C. Cells were spread onto LB agar plates with ampicillin (200 µg/ mL) and incubated for 16 h at 37 °C. A positive transformant was selected and used to inoculate an overnight starter culture (30 mL) of LB medium with ampicillin (200 µg/mL) and 1% glucose. The next day, cells were harvested by centrifugation (5000g, 5 min) and resuspended to an OD equal to 0.01 in Terrific Broth (2L) containing ampicillin (200 µg/ mL). The culture was incubated for several hours (37 °C, 250 rpm) until the OD reached 0.6-0.8. At that time, the inducer (either 1 mM IPTG or 1% lactose) was added to the culture to initiate recombinant protein production. Cells were cultured for an additional 3 h and then harvested by centrifugation (5000g, 5 min). The supernatant was decanted, and the remaining cell pellets were stored at -70 °C. Protein Purification. Frozen cell pellets were thawed for 10 min and resuspended in lysis buffer (10 mL/g wet cell wt., containing 100 mM NaH2PO4, 10 mM Tris-HCl, and 1 mM PMSF, pH 8) for 30 min in a 30 °C shaker. Subsequently, MgCl2 (10 mM), DNAse I (20 mg/mL), and RNase A (20 mg/mL) were added to the resuspended cell lysate and incubated for an additional 30 min in a 30 °C shaker. Cells were placed on ice and sonicated three to four times using a Branson Sonifier (model 450, 30% duty, power level 3). Guanidine-HCl (6 M) and imidazole (5 mM) were added to the cell lysate, mixed thoroughly, and centrifuged at 10000g for 30 min. The soluble protein fraction was filtered (0.45 um) and loaded (150 mL) directly onto a nickelchelated sepharose column (10 mL) equilibrated with 10 column volumes of denaturing buffer (8 M urea, 100 mM NaH2PO4, 10 mM Tris-HCl, 5 mM imidazole, pH 8). Using the AKTA FPLC, the target protein was purified under denaturing conditions with a 20-200 mM imidazole elution gradient. Eluted protein fractions were analyzed by SDSPAGE (4-12% acrylamide, Tris-glycine gel) and silver staining. Pure fractions were pooled and concentrated 10fold via ultrafiltration using an Amicon stirred cell (model 8200). Concentrated protein samples were dialyzed into 2% acetic acid (dialysis tubing, 3.5 kDa MWCO) and lyophilized for long-term storage (yield ∼ 5 mg/L culture). Results and Discussion Gene Construction. The amino acid composition, sequence, and molecular weight of the protein polymers were directly controlled by the DNA sequence and length of the synthetic gene. The DNA monomer encoding the “EAK” motif was constructed from chemically synthesized oligonucleotides that were designed to satisfy E. coli’s codon bias and to avoid strict periodicity in codon usage (Figure 1). Annealed oligonucleotides and the recipient plasmid, pBluescript SKII, were both treated with the restriction endonucleases Bam HI and EcoR I, ligated, and used to transform the recA-deficient E. coli strain, DH10B. Recombinant plasmids were identified by blue/white screening and the monomer DNA sequence was confirmed by dideoxy sequencing using the T3 and T7 universal primers.

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Figure 2. Cleavage of a synthetic DNA duplex flanked by inverted Eam1104 I recognition sites.

Figure 3. Concatemerization of DNA monomers, 4-20% PAGE. Lanes: 1, DNA molecular weight standard (Bio-RAD); 2, DNA monomer; 3, DNA multimers.

Large quantities (∼500 µg) of the monomer DNA were generated by amplification of the recombinant plasmid in an overnight culture (500 mL) and purified using the Qiagen Maxiprep procedure (Qiagen Inc.). The monomer DNA was cleaved from pBluescript SK II at flanking and inverted recognition sites for the type IIs restriction endonuclease, Eam1104 I (Figure 2). The use of Eam1104 I in generating seamless junctions for directional cloning has been reported previously.21,25 The method relies on the ability of type IIs restriction endonucleases to cut downstream of their recognition sequence. DNA monomers cleaved with Eam1104 I generate 5′ cohesive ends in which the three base overhang is independent of the recognition site. Following treatment with Eam1104 I, DNA monomers were purified by gel electrophoresis and enzymatically multimerized by T4 DNA ligase. The degree of polymerization was analyzed by gel electrophoresis (Figure 3) and was controlled by varying the reaction time and the DNA/ ligase concentration. DNA multimers up to 2-3 Kb in length were routinely obtained from self-ligation of monomer DNA (48 bp in length). These higher order multimers were purified by gel extraction prior to ligation with the recipient plasmid. The plasmid pET-19b (Novagen) was selected for inducible protein production. When using pET vectors, transcription of the artificial coding sequence is driven by the T7 phage RNA polymerase. Advantages of the pET system include high bacteriophage T7 transcription levels, and the addition of N-terminal and/or C-terminal fusion tags that facilitate protein detection and purification. The plasmid pET19b also encodes an N-terminal 10 × histidine tag for use in protein purification by immobilized metal affinity chromatography. To prepare pET-19b for cloning, the internal Sap I site was first removed by site directed mutagenesis. Sap I is similar to Eam1104 I in that it is a Type IIs restriction endonuclease that will cleave downstream of the recognition sequence, thereby generating overhangs compatible to the DNA multimers without incorporating extraneous nucleotides. Mutant oligonucleotide primers, each compatible to

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complementary strands of the pET-19b vector, were extended during temperature cycling with PFU Turbo polymerase (Stratagene). Following temperature cycling, the PCR product was treated with Dpn I, a restriction endonuclease specific for methylated and hemimethylated DNA, resulting in digestion of the parent DNA and selection of the mutationcontaining synthesized DNA. Plasmid DNA isolated from most E. coli strains is dam methylated and therefore subject to cleavage by Dpn I digestion. The treated PCR product was used to transform the E. coli strain TOP10F′, and the resulting clones were selected by antibiotic resistance and screened by Sap I digestion to identify mutants of the pET19b vector. To make the mutant plasmid ligation competent, plasmid DNA was amplified by the inverse PCR method26 using primers that included Sap I restriction sites. During the inverse PCR reaction, primers containing Sap I sites were designed to anneal in the opposite orientation to those normally employed in PCR in order to amplify the entire mutant pET-19b (excluding a portion of the multiple cloning site). The result was a linear vector with termini containing Sap I recognition sites that were designed to be compatible with the DNA multimers. Cloning and High-Throughput Screening of DNA Multimers. Purified inverse PCR product was digested with Sap I and treated with calf intestinal alkaline phosphatase to reduce intrachain recircularization during the subsequent ligation reaction. Dephosphorylated plasmid DNA was enzymatically ligated to the DNA multimers using T4 DNA ligase. Recombinant plasmids were initially transformed using the E. coli strain DH10B, which lacks the gene for T7 RNA polymerase. The purpose of this strategy was to eliminate any plasmid instability due to the production of proteins potentially toxic to the host cell. Once established in the nonexpression host, recombinant plasmids were purified and screened for the presence and size of the insert by treatment with Nde I and BamH I. The use of 96-well plates for high-throughput screening proved key in the isolation of plasmids containing highmolecular weight multimers. Positive transformants were incubated overnight in 96-deep well plates containing 2xYT medium (1 mL) with ampicillin. Cells were harvested by centrifugation using a microplate rotor, and the plasmid DNA was purified via vacuum filtration using multiscreen filtration plates (Millipore). Purified plasmid DNA was transferred to 96-well, V-bottom plates and digested with the restriction endonucleases Nde I and Hind III. Samples were analyzed by horizontal gel electrophoresis using Bio-Rad’s model 192 Sub-Cell (100 wells). Plasmids containing higher molecular weight inserts were identified by restriction fragment analysis and confirmed by dideoxy sequencing with both forward and reverse primers. High-throughput screening identified several potential expression vectors containing higher order multimers. They ranged from pET19-EAK9 with nine repeats of the 48 bp DNA monomer to pET19-EAK63 with approximately 63 DNA monomers. The assembled genes correspond to Histagged poly-EAK fusion proteins ranging in size from 17.4 kDa (pET19-EAK9) to 104 kDa (pET19-EAK63).

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Figure 4. Protein expression and purification of poly-EAK9, 4-12% SDS-PAGE, silver stained. Lanes 1-7: 1, protein molecular weight standard; 2-3, E. coli BL21 (DE3) controls (2, noninduced, and 3, with 1 mM IPTG); 4-7, pET19-EAK9, BL21(DE3) (4, noninduced, 5, with 1 mM IPTG, 6, 80 mM imidazole fraction, 7, purified poly-EAK9 in 200 mM imidazole elution fraction).

Protein Production. The E. coli strain BL21(DE3) was selected for recombinant protein production. In strains containing the λDE3 lysogen, a gene encoding T7 RNA polymerase is incorporated into the bacterial chromosome under control of the lacUV5 promoter, providing inducible gene expression following the addition of IPTG. After the recombinant plasmids were established in the host strain, expression of the fusion proteins was induced by adding IPTG to small growing cultures. To detect protein expression levels, aliquots were removed at various time points and analyzed by SDS-polyacrylamide gel electrophoresis (SDSPAGE). Proteins were visualized either by silver staining or by radioactive labeling via incorporation of [S35]-methionine into the N-terminal fusion tag of the protein polymer. From these studies, the plasmid pET19-EAK9 was found to produce the highest levels of target protein (data not shown), and was therefore selected for further production scale-up and characterization. The protein expression studies were also used to determine the optimum IPTG concentration and induction period to maximize protein yield. Large-Scale Protein Production and Purification. Expression from the plasmid pET19-EAK9 affords a translational fusion of the target protein with an N-terminal 10 × histidine tag. The presence of this oligopeptide tag provides a convenient method of protein purification using immobilized metal affinity chromatography (IMAC). The recombinant expression strain was grown to midlog phase in Terrific Broth medium (2-9 L), and target protein production was induced by the addition of 1 mM IPTG or 1% lactose. Cells were harvested after a 3-h induction period and purified under denaturing conditions using an IMAC column with a 20-200 mM imidazole elution gradient. Fractions of purified poly-EAK9 eluted with 200 mM imidazole display a high degree of purity as visualized by SDS-PAGE and silver staining (Figure 4). Characterization. The identity of the purified fusion protein poly-EAK9 was confirmed by amino acid analysis, N-terminal protein sequencing, and MALDI-TOF mass spectrometry (theoretical 17236.0 m/z; experimental 17237.3 m/z). The secondary structure of poly-EAK9 in water at 25 °C was measured by far-ultraviolet circular dichroism (CD) spectroscopy (Figure 5). A range of protein concentrations was examined, each exhibiting a minimum in the CD signal

Improved Assembly of Multimeric Genes

Figure 5. Determination of poly-EAK9’s β-sheet structure in water at 25 °C. CD spectra measured at varying protein concentrations: (9) 2.5 µM, (b) 1.25 µM, and (2) 0.625 µM.

at 218 nm and a maximum signal around 192 nm, characteristic of a β-sheet protein. The β-sheet structure appears independent of protein concentration and is stable even at low concentrations, as indicated by the isosbestic point at 205 nm. Poly-EAK9’s resistance to denaturation over a range of pH, temperature, and urea concentrations along with its self-assembling properties will be reported in a subsequent communication.27 Conclusions Unlike solid-state peptide synthesis and other synthetic chemistries, the use of a bacterial expression system provides unique control over the molecular weight and amino acid sequence of a de novo designed protein polymer. With improved recombinant DNA techniques and high-throughput screening methods, this approach should prove useful in the development of novel protein polymers encoding repetitive amino acid sequences. From these methods, we have constructed a high molecular weight analogue of the EAK sequence, poly-EAK9, and expressed and purified the artificial protein from E. coli. Current studies are underway to probe poly-EAK9’s self-assembly and mechanical properties for potential use as a biomaterial. Acknowledgment. We thank Professor Susan Marqusee for the use of the AVIV 62DS spectrometer. This work was supported by the UC Biotechnology Program, the National Science Foundation (BES-0103468), and an NSF Graduate Fellowship (N.L.G-W.). References and Notes (1) van Hest, J.; Tirrell, D. A. Protein-based materials, toward a new level of structural control. Chem. Commun. 2001, 19, 1897-1904. (2) Heslot, H. Artificial fibrous proteins: A review. Biochimie 1998, 80 (1), 19-31. (3) O’Brien, J. P.; Fahnestock, S. R.; Termonia, Y.; Gardner, K. C. H. Nylons from nature: Synthetic analogues to spider silk. AdV. Mater. 1998, 10, 1185. (4) Protein-Based Materials; McGrath, K., Kaplan, D. Ed.; Birkhauser: Boston, MA, 1997. (5) Zhou, Y. T.; Wu, S. X.; Conticello, V. P. Genetically directed synthesis and spectroscopic analysis of a protein polymer derived from a flagelliform silk sequence. Biomacromolecules 2001, 2, 111125. (6) McMillan, R. A.; Conticello, V. P. Synthesis and characterization of elastin-mimetic protein gels derived from a well-defined polypeptide precursor. Macromolecules 2000, 33, 4809-4821.

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