- Email: [email protected]
Ribosomal synthesis and in situ isolation of peptide molecules in a cell-free translation system
Protein Expression and Purification 71 (2010) 16–20
Contents lists available at ScienceDirect
Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
Ribosomal synthesis and in situ isolation of peptide molecules in a cell-free translation system Kyung-Ho Lee a, Yong-Chan Kwon b, Sung Joon Yoo c, Dong-Myung Kim a,b,* a
Interdisciplinary Program for Nano-Technology, Chungnam National University, Daejeon 305-764, Republic of Korea Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764, Republic of Korea c Bioshield Co. Ltd., Rm 3118, HTVC KAIST 373-1, 373-1 Gusung-dong, Daejeon 305-701, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 22 September 2009 and in revised form 8 January 2010 Available online 25 January 2010 Keywords: Peptides Cell-free translation Downstream box Chloramphenicol acetyltransferase Magainin Opistoporin Human b-defensin-2 Tachystatin A
a b s t r a c t Although the cell-free translation system is now widely accepted as an efficient platform for production, engineering and screening of recombinant proteins, it has not been successfully used for the synthesis of peptide molecules mainly due to low expression yields and rapid proteolysis of the expressed peptides. In this study, we propose a novel strategy for rapid expression and recovery of peptide molecules which involves the rational design of template DNA and heterogenous cell-free translation reaction in the presence of affinity beads. Various peptide molecules which were not expressed in a detectable level were successfully expressed and recovered in situ in a substantial yield. We expect that the presented approach will be widely used as a versatile platform for the generation of a variety of peptide molecules. Ó 2010 Elsevier Inc. All rights reserved.
Introduction Depending on their amino acid sequences, peptide molecules exhibit extraordinarily diverse biological functions ranging from hormonal regulation to antibiotic activities, and thus are of paramount importance in pharmaceutical fields. In 2004, there were more than 40 therapeutic peptides on the market [1], and now a much larger number of peptides are in different phases of clinical trials or are awaiting approval for clinical use. Peptide molecules also have potential use in the field of nano-biotechnology. Their unique properties, such as binding affinity to specific metals [2] or the formation of self-assembled structures [3], can enrich the arsenal of materials available for fabrication of nano-sensors or other nano-structured devices. Exploration of the huge sequence space of peptides can be accelerated using methods that enable the generation and analysis of peptides in a high-throughput manner. While peptides can be produced by chemical synthesis or in vivo expression of corresponding nucleotide sequences, both methods have substantial drawbacks to be used for rapid generation of multiple peptides.
* Corresponding author. Address: Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764, Republic of Korea. Fax: +82 42 823 7692. E-mail address: [email protected] (D.-M. Kim). 1046-5928/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2010.01.016
In general, chemical synthesis of peptides is time-consuming and costly [4,5] mainly due to the requirement for repeated blocking and deblocking steps, and the complexity of chemical peptide synthesis increases in proportion to the number of amino acids. On the other hand, while the cell-based expression method can be used for peptide production at cheaper costs, the requirement for time- and labor intensive cloning and cell cultivation procedures limits its use for high-throughput generation of peptide molecules. It often suffers low expression yield and/or proteolytic degradation of the translation products. In particular, those peptides that exert toxic effect on the host cells cannot be produced in living cells. Cell-free translation techniques can be used as an alternative way to address the problems associated with in vivo and chemical synthesis methods. Through the use of translation machinery isolated from the cells, peptide molecules can be directly obtained from DNA or RNA templates without involving the complicated protection and deprotection steps that are required for chemical synthesis. At the same time, compared to the in vivo expression methods, cell-free synthesis of peptides brings substantial benefits with respect to the throughputs of peptide generation. In addition to that cell cultivation steps can be bypassed, cell-free synthesis reactions can be carried out using PCR-amplified DNAs as the direct template for peptide expression, thereby avoiding trivial cloning procedures. Moreover, since the utilization of translational
K.-H. Lee et al. / Protein Expression and Purification 71 (2010) 16–20
machinery is uncoupled from the growth of the cells, cell-free synthesis system can provide an ideal platform for the expression of cytotoxic products such as antimicrobial peptides (AMPs1). Although conventional cell-free translation technology has long suffered from an unacceptably low productivity and high reagent costs, recent advances in this field have resulted in remarkable improvements in the efficiency and economics of cell-free protein synthesis [6,7]. For example, through the enhanced protein productivity using cheaper energy sources, it was recently shown that a milligram of protein can be produced for less than 0.5 US dollars using a cell-free translation system [8,9]. Nevertheless, cell-free translation systems optimized for protein production do not necessarily provide sufficient quantities of the desired peptide. As was discussed previously by Stephanopoulos and co-workers, short peptides are apt to be digested by proteases present in the cell extract, and the susceptibility to proteolytic digestion tends to be inversely proportional to their lengths [10]. In addition to the rapid proteolytic digestion in the cell extract, we also speculate that poor efficiency of translation would be at least partially responsible for the insufficient accumulation of peptide molecules during cell-free translation reactions. In this study, we demonstrate that purified peptides can be produced in remarkably improved yields through rational design of the primers to prepare the template DNAs for their expression. Designed primers include the sequences for enhancing the translational efficiency and resistance against proteolytic digestion, in situ separation of the translation products onto IMAC resins, and recovery of peptides having authentic sequences. Materials and methods Materials ATP, GTP, UTP, CTP, creatine phosphate (CP), and creatine kinase (CK) were purchased from Roche Applied Science (Indianapolis, IN). All other reagents were purchased from Sigma and used without further purifications. The S12 extract from BL21(DE3)-star was prepared as described previously after minor modifications [11]. Instead of its exogenous addition, the T 7 RNA polymerase was expressed during the cultivation of Escherichia coli with isopropyl-D-thiogalactoside induction (1.0 mM) at 0.5 OD600. The cells were harvested 2 h after induction and used to prepare the S12 extract.
17
Fig. 1. (A) Schematic representation of ribosomal synthesis and in situ isolation of target peptides with designed leading sequence for efficient translation and authentic peptide. (B) Gene construction of the expression-ready templates for cell-free peptide synhtesis.
to include a translation-enhancing sequence upstream of the peptide sequence. The two primary PCRs were conducted under standard conditions as follows: the CAT-DB sequence containing the T 7 promoter and RBS were amplified from pK7 CAT. In addition, the primer added a hexa-histidine sequence between the CAT-DB and target peptide sequence to isolate peptides in situ using immobilized metal affinity chromatography (IMAC) beads in the reaction mixture. Finally, a Factor Xa cleavage site was added immediately next the hexa-histidine sequence to remove the amino acid residues derived from the CAT-DB and hexa-histidine tag after the in situ isolation steps. Next, target ORFs of the target peptide containing the T 7 terminator were amplified from each pK7 vector coding peptide. The PCR products were purified by gel extraction and used for the second-round PCR, in which the full-length expression templates were synthesized (Fig. 2). After amplification, the PCR products were purified using a PCR clean up kit (Promega, Madison, WI) before being used in the cell-free translation reactions. Cell-free translation and in situ isolation of peptides
Construction of wild-type peptide templates Four antimicrobial peptides, magainin 2 (ma2) [12], opistoporin 1 (opi1) [13], human b-defensin-2 (hbd2) [14], and tachystatin A1 (tacA1) [15] were chosen as target peptides for cell-free translation and in situ isolation. Gene fragments were designed using GeneDesign b2.0 (http://baderlab.bme.jhu.edu/gd/), and the synthesized oligonucleotides (Supplementary Table S1) were assembled in a two step PCR reaction to construct the structural genes of the peptides flanking the NdeI/SalI restriction sites. After digestion with NdeI and SalI, the amplified genes were cloned into the pK7 plasmid [16].
The standard reaction mixture for the cell-free translation of the PCR-amplified template DNAs consisted of the following components at a final volume of 15 ll; 57 mM Hepes–KOH (pH 7.5),
Preparation of PCR-amplified genes for CAT-DB fusion peptides CAT-DB fused-gene constructs (Fig. 1A) were prepared via overlap PCR reactions (Supplementary Table S2). In the first-round of PCR, the primer for amplification of the peptide gene was designed 1 Abbreviations used: AMPs, antimicrobial peptides; CP, creatine phosphate; CK, creatine kinase; ma2, magainin 2; opi1, opistoporin 1; hbd2, human b-defensin-2; tacA1, tachystatin A1; IMAC, immobilized metal affinity chromatography.
Fig. 2. Cell-free synthesis of peptide molecules. Lane 1: molecular weight markers; 2: control reaction without DNA; 3–6: reactions programmed with the peptide genes (ma2, opi1, hbd2 and tacA, respectively).
18
K.-H. Lee et al. / Protein Expression and Purification 71 (2010) 16–20
1.2 mM ATP, 0.85 mM each GTP, UTP, and CTP, 80 mM ammonium acetate, 34 lg/ml 1-5-formyl-5,6,7,8-tetrahydrofolic acid (folinic acid), 2.0 mM each of 20 amino acids, 0.3 U/ml creatine kinase, 67 mM creatine phosphate, 0.5 lg PCR-amplified DNA, and 4 ll of the S12 extract. In the experiments for in situ isolation of peptides (Fig. 1B), cell-free translation reactions were carried out in the presence of 15 ll of 5% Ni–NTA equilibrated agarose magnetic beads (QIAGEN, Hilden, Germany). After 2 h of incubation, the magnetic beads were recovered using a magnet and transferred to 15 ll of 20 mM Tris–HCl (pH 8.0) buffer containing 0.2 lg of Factor Xa (New England Biolabs, MA). Synthesized peptides were analyzed by Coomassie Blue staining after running the reaction samples on a 16% Tricine–SDS–polyacrylamide gel with 6 M urea as described by Schagger [17]. Results and discussion Inefficient synthesis of wild-type peptides in a cell-free translation system When the reaction mixture for cell-free translation was programmed with 4 different peptide genes, magainin 2, opistoporin 1, human b-defensin-2, and tachystatin A1 prepared by PCR reactions, most of the peptides were not detected on a Tricine–SDS– PAGE gel stained with Coomassie Brilliant Blue (Fig. 2). However, cell-free expression of the opi1 gene was an exception, and gave a clear and strong band on the Tricine–PAGE gel. Since the mRNA level in the reaction mixture was found to be similar among all of the examined genes (data not shown), it appeared that the difference in the amounts of accumulated peptides resulted from variations in the translational efficiency and/or stability against proteases present in the cell extract. In addition, since the size of opistoporin 1 is not substantially larger than the others, the efficiency of peptide accumulation did not seem be determined simply by their lengths. Enhanced synthesis of peptides with designed leading sequences in a cell-free translation system There have been many publications reporting that the efficiency of gene expression is dramatically affected by the nature of the initial nucleotide sequences immediately next to the start codon. For instance, it was shown that the translation efficiency of a lacZ reporter gene could be varied as much as 20-fold depending upon the codon at +2 position [18]. In addition, we also found in our previous study that the translational efficiency of mRNAs became highly diversified through the randomization of their initial codons [19]. Based on these findings indicating that the efficiency of gene expression is primarily determined during the early stage of translation, we thought we might be able to enhance the translation efficiency of peptide genes by placing ‘good’ codons in front. For this purpose, PCR primers were designed to add the initial codons of chloramphenicol acetyltransferase (CAT) which was one of the most efficiently expressed proteins in our system [16], to the up-
stream of the nucleotide sequence of the target peptides. The primers were also designed to contain a hexa-histidine sequence between the CAT-derived sequence (CAT-DB) and the target peptide sequence to enable in situ isolation of the expressed peptides using immobilized metal affinity chromatography (IMAC) beads in the reaction mixture. Expression-ready genes were prepared through the 2 step PCR reactions. Finally, an enzyme cleavage site for Factor Xa was introduced immediately next to the hexa-histidine sequence to enable the removal of the auxillary amino acid residues and recovery of the peptides of the native sequence after the in situ isolation steps (Fig. 1). Indeed, it was observed that the addition of these auxiliary sequences remarkably enhanced the yield of peptide synthesis during the cell-free synthesis reactions. These results indicate that short peptides can be effectively expressed in a cell-free translation system when the efficiency of translation was enhanced by placing a ‘booster sequence’ in front of the target peptides that are otherwise poorly expressed. The presence of the CAT-DB sequences led to the successful expression of three peptides (magainin 2, human b-defensin-2 and tachystatin A1) in sufficient amounts to be observed on a Coomassie Blue-stained gel (Fig. 3). From the relative intensity of the bands, the CAT-DB sequences corresponding to the initial 10 (CAT-DB10) to 13 (CAT-DB13) amino acids appear to be optimal for the expression of these peptides. Densitometric analysis of the bands indicated that approximately 80 (magainin 2), 450 (opistoporin), 360 (human b-defensin-2) and 120 (tachystatin A1) lg/ml of the fusion peptides were produced. Interestingly, when we introduced different silent mutations to the CAT-DB sequence, the intensity of the bands were substantially diminished (data not shown), indicating that the effect of the CAT-DB sequence addition was at least partially due to the enhanced translational efficiency by the nucleotide sequences of the CAT-DB. In order to determine if the effect of the CAT-DB sequences was due to the presence of additional nucleotide sequences or the resulting amino acid residues, indicating that the effect of the CAT-DB sequence addition was at least partially due to the enhanced translational efficiency by the nucleotide sequences of the CAT-DB. Effect of the leader sequence on the stability of cell-free synthesized peptides Short peptides are generally considered to be highly susceptible to proteolytic degradation in the cytoplasm of bacterial cells and their extracts, which was also confirmed in our experiment using chemically synthesized Ma2 as a model peptide. When Ma2 (300 lg/ml) of its authentic sequence was incubated in the same cell extract used for cell-free translation, the peptide was degraded almost completely within an hour (Fig. 4A, lane 4). However, no notable degradation was observed in the heat-treated (5 min at 80 °C) cell extract (Fig. 4A, lane 6) indicating that the endogenous proteases in the cell extract were responsible for the rapid degra-
Fig. 3. Cell-free expression of peptides with the initial codons of chloramphenicol acetyltransferase (CAT-DB) of variable lengths (number of amino acid residues from the CAT-DB are indicated).
K.-H. Lee et al. / Protein Expression and Purification 71 (2010) 16–20
19
the fusion partner, 20-fold more Ma2 (2.5 kDa) will be recovered when the same molarity of the translation product is produced. This is of particular importance in the cell-free translation systems where the synthesis of peptides is carried out using limited pool of resources including the energy sources and amino acids. In addition, since no cysteine residues are included in this fusion sequence, it would not interfere with the correct formation of disulfide bonds within the target peptides containing multiple cysteine residues. Recovery of authentic peptide molecules from in situ immobilized peptide-magnetic beads
Fig. 4. Protective effect of the CAT-DB sequences. (A) Degradation of synthetic peptide Ma2 in the cell extract. Lane 1: markers; 2: only synthetic MA2; 3: cell extracts; 4: Ma2 incubated in the cell extract for 1 h; 5: Ma2 incubated in the cell extract in the presence of protease inhibitor cocktail; 6: MA2 incubated in the heattreated cell-extracts; 7: cell-free translation of cat(10)-db ma2 during 2 h; 8: cellfree translation of cat(10)db-ma2 during 2 h and additive incubation for 1 h after the addition of chloramphenicol. (B) Degradation of native hBD2 by FXa-mediated in situ cleavage. Lane 1: markers; 2: negative control without DNA; 3: incubation of cell-free mixture with FXa; 4: cell-free translation of cat(10)db-hbd2; 5: Cell-free translation of cat(10)db-hbd2 with FXa.
dation of the peptide. Interestingly, unlike the reported results of Stephanopoulos, addition of a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) was not effective in preventing the degradation of Ma2 (Fig. 4A, lane 5). In contrast to the authentic sequence of Ma2, the translation product from the fusion construct of CAT-DB-Ma2 appeared to be highly resistant to the proteolytic digestion. After two hours of the cell-free translation reaction of cat(10)-db ma2, chloramphenicol (170 lg/ml of final concentration) was added to the reaction mixture to halt peptide synthesis. The reaction mixture was further incubated for an additional hour, and then analyzed on a Tricine– PAGE gel. As shown in Fig. 4A, the CAT-DB fused peptide maintained its intensity undeteriorated at least for 3 h. Therefore, in addition to the stimulatory effect on translation, it also appears that CAT-DB sequences improve the stability of the peptides against proteolytic digestion. The protective effect of the CAT-DB sequence does not seem to be limited to Ma2. For instance, when the hbd2 sequence fused with cat(10)-db was expressed in the cell-free translation system, the intensity of the Coomassie Blue-stained band of the translation product remained unchanged over 3 h. However, upon the removal of the auxiliary sequence (CAT-DB and hexa-histidine) by the addition of Factor Xa, instant degradation of the peptide was observed (Fig. 4B, lane 5), supporting the hypothesis that the presence of these auxiliary sequences plays an important role in protecting the peptide molecules. Taken together, we concluded that the CAT-DB sequences serve as an effective fusion partner for highyield ribosomal synthesis of peptides by both enhancing translational efficiency and protecting against proteolytic digestion. Although a number proteins have been used as the fusion partners to express peptide molecules [20], the fusion sequence designed in this study (Supplementary Table S3) offers substantial advantages compared to those conventional fusion partners. First, due to its small size (1.7–2.8 kDa), more transcriptional/translational resources can be directed for the synthesis of target peptides. For example, compared to the use of NusA (54.9 kDa) as
The open nature of cell-free translation also offers a unique advantage in the recovery of the expressed peptides. As depicted in the scheme of Fig. 1, through the expression of the designed gene constructs in the presence of affinity beads, we attempted to recover the translation product instantly after the synthesis reaction. As an example of this approach of in situ isolation, the cat-db-hexa-histidine-hbd2 construct was incubated in the reaction mixture containing Ni–NTA magnetic agarose beads. After 2 h of incubation, the magnetic beads were recovered by a magnet, washed, and then incubated in a buffer solution containing 13 lg/ml of Factor Xa for 1 h at 37 °C. When the supernatant was run on a 16% Tricine gel containing 6 M urea, a clear peptide band was observed at the expected size for human b-defensin (Fig. 5), indicating that the native peptide was successfully released from the bead leaving the auxiliary amino acid residues (CAT-DB and hexa-histidine tag) bound on the beads. It was estimated that approximately 80 lg of hBD2 was recovered from 1 ml of cell-free translation reaction mixture. Conclusion We demonstrated that the addition of properly designed leader sequences remarkably improves the productivity and convenience of peptide synthesis during cell-free translation reactions. The ability to use PCR-amplified genes as the direct template for cell-free peptide synthesis brings substantial benefits in the throughput of peptide production, compared to the conventional chemical synthesis and in vivo expression methods. Although the cell-free synthesis of peptides generally suffers from extremely low productivity, we found that the use of short CAT-DB sequence could address the issues of inefficient translation and proteolytic digestion of the expressed peptides. In addition, through the introduction of affinity tag sequence and enzyme cleavage site between the CAT-DB and target peptide sequence, target peptides of authentic amino acid sequence were easily recovered from the reaction mixture. Cost analysis of cell-free peptide synthesis indicates that the approach of cell-free synthesis can also be economically viable. As shown in Fig. 6, based on the catalogue prices of the reagents, approximately 4 USD are required for the
Fig. 5. In situ isolation of hBD2 following FXa reaction for authentic hBD2. Lane 1: markers; 2: in situ isolated CAT(10)-DB hBD2 without FXa reaction; 3: authentic hDB2 after FXa reaction.
20
K.-H. Lee et al. / Protein Expression and Purification 71 (2010) 16–20
Fig. 6. Reagent costs for a 1 ml reaction and 1 mg peptide synthesis in the cell-free system.
expression of 1 mg of hBD2. Although purification costs are not included in this estimation, considering the commercial prices of chemically synthesized peptides (approximately 100 USD for the sequence of hBD2), cell-free synthesis appears to be a feasible option at least for the generation of peptides consisted of natural amino acids. Acknowledgments Authors gratefully acknowledge financial support from the Small and Medium Business Administration of Korea (Grant No. S1033489). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.pep.2010.01.016. References [1] F. Albericio, Developments in peptide and amide synthesis, Current Opinion in Chemical Biology 8 (2004) 211–221. [2] C. Tamerler, M. Duman, E.E. Oren, M. Gungormus, X.R. Xiong, T. Kacar, B.A. Parviz, M. Sarikaya, Materials specificity and directed assembly of a goldbinding peptide, Small 2 (2006) 1372–1378.
[3] K. Rajagopal, J.P. Schneider, Self-assembling peptides and proteins for nanotechnological applications, Current Opinion in Structural Biology 14 (2004) 480–486. [4] P.W. Latham, Therapeutic peptides revisited, Nature Biotechnology 17 (1999) 755–757. [5] M. Amblard, J.A. Fehrentz, J. Martinez, G. Subra, Fundamentals of modern peptide synthesis, Methods in Molecular Biology 298 (2005) 3–24. [6] T.W. Kim, I.S. Oh, J.W. Keum, Y.C. Kwon, J.Y. Byun, K.H. Lee, C.Y. Choi, D.M. Kim, Prolonged cell-free protein synthesis using dual energy sources: combined use of creatine phosphate and glucose for the efficient supply of ATP and retarded accumulation of phosphate, Biotechnology and Bioengineering 97 (2007) 1510–1515. [7] A.R. Goerke, J.R. Swartz, High-level cell-free synthesis yields of proteins containing site-specific non-natural amino acids, Biotechnology and Bioengineering 102 (2009) 400–416. [8] T.W. Kim, J.W. Keum, I.S. Oh, C.Y. Choi, H.C. Kim, D.M. Kim, An economical and highly productive cell-free protein synthesis system utilizing fructose-1,6bisphosphate as an energy source, Journal of Biotechnology 130 (2007) 389– 393. [9] T.W. Kim, H.C. Kim, I.S. Oh, D.M. Kim, A highly efficient and economical cellfree protein synthesis system using the S12 extract of Escherichia coli, Biotechnology and Bioprocess Engineering 13 (2008) 464–469. [10] C.R. Loose, R.S. Langer, G.N. Stephanopoulos, Optimization of protein fusion partner length for maximizing in vitro translation of peptides, Biotechnology Progress 23 (2007) 444–451. [11] T.W. Kim, J.W. Keum, I.S. Oh, C.Y. Choi, C.G. Park, D.M. Kim, Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system, Journal of Biotechnology 126 (2006) 554–561. [12] M. Zasloff, Magainins, a class of antimicrobial peptides from Xenopus skin – Isolation, characterization of 2 active forms, and partial cDNA sequence of a precursor, Proceedings of the National Academy of Sciences of the United States of America 84 (1987) 5449–5453. [13] L. Moerman, S. Bosteels, W. Noppe, J. Willems, E. Clynen, L. Schoofs, K. Thevissen, J. Tytgat, J. Van Eldere, J. van der Walt, F. Verdonck, Antibacterial and antifungal properties of alpha-helical, cationic peptides in the venom of scorpions from southern Africa, European Journal of Biochemistry 269 (2002) 4799–4810. [14] D.M. Hoover, K.R. Rajashankar, R. Blumenthal, A. Puri, J.J. Oppenheim, O. Chertov, J. Lubkowski, The structure of human beta-defensin-2 shows evidence of higher order oligomerization, Journal of Biological Chemistry 275 (2000) 32911–32918. [15] N. Fujitani, S. Kawabata, T. Osaki, Y. Kumaki, M. Demura, K. Nitta, K. Kawano, Structure of the antimicrobial peptide tachystatin A, Journal of Biological Chemistry 277 (2002) 23651–23657. [16] J.M. Son, J.H. Ahn, M.Y. Hwang, C.G. Park, C.Y. Choi, D.M. Kim, Enhancing the efficiency of cell-free protein synthesis through the polymerase-chainreaction-based addition of a translation enhancer sequence and the in situ removal of the extra amino acid residues, Analytical Biochemistry 351 (2006) 187–192. [17] H. Schagger, Tricine–SDS–PAGE, Nature Protocols 1 (2006) 16–22. [18] C.M. Stenstrom, L.A. Isaksson, Influences on translation initiation and early elongation by the messenger RNA region flanking the initiation codon at the 30 side, Gene 288 (2002) 1–8. [19] J.H. Ahn, J.W. Keum, D.M. Kim, High-throughput, combinatorial engineering of initial codons for tunable expression of recombinant proteins, Journal of Proteome Research 7 (2008) 2107–2113. [20] J. Bogomolovas, B. Simon, M. Sattler, G. Stier, Screening of fusion partners for high yield expression and purification of bioactive viscotoxins, Protein Expression and Purification 64 (2009) 16–23.
Contents lists available at ScienceDirect
Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
Ribosomal synthesis and in situ isolation of peptide molecules in a cell-free translation system Kyung-Ho Lee a, Yong-Chan Kwon b, Sung Joon Yoo c, Dong-Myung Kim a,b,* a
Interdisciplinary Program for Nano-Technology, Chungnam National University, Daejeon 305-764, Republic of Korea Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764, Republic of Korea c Bioshield Co. Ltd., Rm 3118, HTVC KAIST 373-1, 373-1 Gusung-dong, Daejeon 305-701, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 22 September 2009 and in revised form 8 January 2010 Available online 25 January 2010 Keywords: Peptides Cell-free translation Downstream box Chloramphenicol acetyltransferase Magainin Opistoporin Human b-defensin-2 Tachystatin A
a b s t r a c t Although the cell-free translation system is now widely accepted as an efficient platform for production, engineering and screening of recombinant proteins, it has not been successfully used for the synthesis of peptide molecules mainly due to low expression yields and rapid proteolysis of the expressed peptides. In this study, we propose a novel strategy for rapid expression and recovery of peptide molecules which involves the rational design of template DNA and heterogenous cell-free translation reaction in the presence of affinity beads. Various peptide molecules which were not expressed in a detectable level were successfully expressed and recovered in situ in a substantial yield. We expect that the presented approach will be widely used as a versatile platform for the generation of a variety of peptide molecules. Ó 2010 Elsevier Inc. All rights reserved.
Introduction Depending on their amino acid sequences, peptide molecules exhibit extraordinarily diverse biological functions ranging from hormonal regulation to antibiotic activities, and thus are of paramount importance in pharmaceutical fields. In 2004, there were more than 40 therapeutic peptides on the market [1], and now a much larger number of peptides are in different phases of clinical trials or are awaiting approval for clinical use. Peptide molecules also have potential use in the field of nano-biotechnology. Their unique properties, such as binding affinity to specific metals [2] or the formation of self-assembled structures [3], can enrich the arsenal of materials available for fabrication of nano-sensors or other nano-structured devices. Exploration of the huge sequence space of peptides can be accelerated using methods that enable the generation and analysis of peptides in a high-throughput manner. While peptides can be produced by chemical synthesis or in vivo expression of corresponding nucleotide sequences, both methods have substantial drawbacks to be used for rapid generation of multiple peptides.
* Corresponding author. Address: Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764, Republic of Korea. Fax: +82 42 823 7692. E-mail address: [email protected] (D.-M. Kim). 1046-5928/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2010.01.016
In general, chemical synthesis of peptides is time-consuming and costly [4,5] mainly due to the requirement for repeated blocking and deblocking steps, and the complexity of chemical peptide synthesis increases in proportion to the number of amino acids. On the other hand, while the cell-based expression method can be used for peptide production at cheaper costs, the requirement for time- and labor intensive cloning and cell cultivation procedures limits its use for high-throughput generation of peptide molecules. It often suffers low expression yield and/or proteolytic degradation of the translation products. In particular, those peptides that exert toxic effect on the host cells cannot be produced in living cells. Cell-free translation techniques can be used as an alternative way to address the problems associated with in vivo and chemical synthesis methods. Through the use of translation machinery isolated from the cells, peptide molecules can be directly obtained from DNA or RNA templates without involving the complicated protection and deprotection steps that are required for chemical synthesis. At the same time, compared to the in vivo expression methods, cell-free synthesis of peptides brings substantial benefits with respect to the throughputs of peptide generation. In addition to that cell cultivation steps can be bypassed, cell-free synthesis reactions can be carried out using PCR-amplified DNAs as the direct template for peptide expression, thereby avoiding trivial cloning procedures. Moreover, since the utilization of translational
K.-H. Lee et al. / Protein Expression and Purification 71 (2010) 16–20
machinery is uncoupled from the growth of the cells, cell-free synthesis system can provide an ideal platform for the expression of cytotoxic products such as antimicrobial peptides (AMPs1). Although conventional cell-free translation technology has long suffered from an unacceptably low productivity and high reagent costs, recent advances in this field have resulted in remarkable improvements in the efficiency and economics of cell-free protein synthesis [6,7]. For example, through the enhanced protein productivity using cheaper energy sources, it was recently shown that a milligram of protein can be produced for less than 0.5 US dollars using a cell-free translation system [8,9]. Nevertheless, cell-free translation systems optimized for protein production do not necessarily provide sufficient quantities of the desired peptide. As was discussed previously by Stephanopoulos and co-workers, short peptides are apt to be digested by proteases present in the cell extract, and the susceptibility to proteolytic digestion tends to be inversely proportional to their lengths [10]. In addition to the rapid proteolytic digestion in the cell extract, we also speculate that poor efficiency of translation would be at least partially responsible for the insufficient accumulation of peptide molecules during cell-free translation reactions. In this study, we demonstrate that purified peptides can be produced in remarkably improved yields through rational design of the primers to prepare the template DNAs for their expression. Designed primers include the sequences for enhancing the translational efficiency and resistance against proteolytic digestion, in situ separation of the translation products onto IMAC resins, and recovery of peptides having authentic sequences. Materials and methods Materials ATP, GTP, UTP, CTP, creatine phosphate (CP), and creatine kinase (CK) were purchased from Roche Applied Science (Indianapolis, IN). All other reagents were purchased from Sigma and used without further purifications. The S12 extract from BL21(DE3)-star was prepared as described previously after minor modifications [11]. Instead of its exogenous addition, the T 7 RNA polymerase was expressed during the cultivation of Escherichia coli with isopropyl-D-thiogalactoside induction (1.0 mM) at 0.5 OD600. The cells were harvested 2 h after induction and used to prepare the S12 extract.
17
Fig. 1. (A) Schematic representation of ribosomal synthesis and in situ isolation of target peptides with designed leading sequence for efficient translation and authentic peptide. (B) Gene construction of the expression-ready templates for cell-free peptide synhtesis.
to include a translation-enhancing sequence upstream of the peptide sequence. The two primary PCRs were conducted under standard conditions as follows: the CAT-DB sequence containing the T 7 promoter and RBS were amplified from pK7 CAT. In addition, the primer added a hexa-histidine sequence between the CAT-DB and target peptide sequence to isolate peptides in situ using immobilized metal affinity chromatography (IMAC) beads in the reaction mixture. Finally, a Factor Xa cleavage site was added immediately next the hexa-histidine sequence to remove the amino acid residues derived from the CAT-DB and hexa-histidine tag after the in situ isolation steps. Next, target ORFs of the target peptide containing the T 7 terminator were amplified from each pK7 vector coding peptide. The PCR products were purified by gel extraction and used for the second-round PCR, in which the full-length expression templates were synthesized (Fig. 2). After amplification, the PCR products were purified using a PCR clean up kit (Promega, Madison, WI) before being used in the cell-free translation reactions. Cell-free translation and in situ isolation of peptides
Construction of wild-type peptide templates Four antimicrobial peptides, magainin 2 (ma2) [12], opistoporin 1 (opi1) [13], human b-defensin-2 (hbd2) [14], and tachystatin A1 (tacA1) [15] were chosen as target peptides for cell-free translation and in situ isolation. Gene fragments were designed using GeneDesign b2.0 (http://baderlab.bme.jhu.edu/gd/), and the synthesized oligonucleotides (Supplementary Table S1) were assembled in a two step PCR reaction to construct the structural genes of the peptides flanking the NdeI/SalI restriction sites. After digestion with NdeI and SalI, the amplified genes were cloned into the pK7 plasmid [16].
The standard reaction mixture for the cell-free translation of the PCR-amplified template DNAs consisted of the following components at a final volume of 15 ll; 57 mM Hepes–KOH (pH 7.5),
Preparation of PCR-amplified genes for CAT-DB fusion peptides CAT-DB fused-gene constructs (Fig. 1A) were prepared via overlap PCR reactions (Supplementary Table S2). In the first-round of PCR, the primer for amplification of the peptide gene was designed 1 Abbreviations used: AMPs, antimicrobial peptides; CP, creatine phosphate; CK, creatine kinase; ma2, magainin 2; opi1, opistoporin 1; hbd2, human b-defensin-2; tacA1, tachystatin A1; IMAC, immobilized metal affinity chromatography.
Fig. 2. Cell-free synthesis of peptide molecules. Lane 1: molecular weight markers; 2: control reaction without DNA; 3–6: reactions programmed with the peptide genes (ma2, opi1, hbd2 and tacA, respectively).
18
K.-H. Lee et al. / Protein Expression and Purification 71 (2010) 16–20
1.2 mM ATP, 0.85 mM each GTP, UTP, and CTP, 80 mM ammonium acetate, 34 lg/ml 1-5-formyl-5,6,7,8-tetrahydrofolic acid (folinic acid), 2.0 mM each of 20 amino acids, 0.3 U/ml creatine kinase, 67 mM creatine phosphate, 0.5 lg PCR-amplified DNA, and 4 ll of the S12 extract. In the experiments for in situ isolation of peptides (Fig. 1B), cell-free translation reactions were carried out in the presence of 15 ll of 5% Ni–NTA equilibrated agarose magnetic beads (QIAGEN, Hilden, Germany). After 2 h of incubation, the magnetic beads were recovered using a magnet and transferred to 15 ll of 20 mM Tris–HCl (pH 8.0) buffer containing 0.2 lg of Factor Xa (New England Biolabs, MA). Synthesized peptides were analyzed by Coomassie Blue staining after running the reaction samples on a 16% Tricine–SDS–polyacrylamide gel with 6 M urea as described by Schagger [17]. Results and discussion Inefficient synthesis of wild-type peptides in a cell-free translation system When the reaction mixture for cell-free translation was programmed with 4 different peptide genes, magainin 2, opistoporin 1, human b-defensin-2, and tachystatin A1 prepared by PCR reactions, most of the peptides were not detected on a Tricine–SDS– PAGE gel stained with Coomassie Brilliant Blue (Fig. 2). However, cell-free expression of the opi1 gene was an exception, and gave a clear and strong band on the Tricine–PAGE gel. Since the mRNA level in the reaction mixture was found to be similar among all of the examined genes (data not shown), it appeared that the difference in the amounts of accumulated peptides resulted from variations in the translational efficiency and/or stability against proteases present in the cell extract. In addition, since the size of opistoporin 1 is not substantially larger than the others, the efficiency of peptide accumulation did not seem be determined simply by their lengths. Enhanced synthesis of peptides with designed leading sequences in a cell-free translation system There have been many publications reporting that the efficiency of gene expression is dramatically affected by the nature of the initial nucleotide sequences immediately next to the start codon. For instance, it was shown that the translation efficiency of a lacZ reporter gene could be varied as much as 20-fold depending upon the codon at +2 position [18]. In addition, we also found in our previous study that the translational efficiency of mRNAs became highly diversified through the randomization of their initial codons [19]. Based on these findings indicating that the efficiency of gene expression is primarily determined during the early stage of translation, we thought we might be able to enhance the translation efficiency of peptide genes by placing ‘good’ codons in front. For this purpose, PCR primers were designed to add the initial codons of chloramphenicol acetyltransferase (CAT) which was one of the most efficiently expressed proteins in our system [16], to the up-
stream of the nucleotide sequence of the target peptides. The primers were also designed to contain a hexa-histidine sequence between the CAT-derived sequence (CAT-DB) and the target peptide sequence to enable in situ isolation of the expressed peptides using immobilized metal affinity chromatography (IMAC) beads in the reaction mixture. Expression-ready genes were prepared through the 2 step PCR reactions. Finally, an enzyme cleavage site for Factor Xa was introduced immediately next to the hexa-histidine sequence to enable the removal of the auxillary amino acid residues and recovery of the peptides of the native sequence after the in situ isolation steps (Fig. 1). Indeed, it was observed that the addition of these auxiliary sequences remarkably enhanced the yield of peptide synthesis during the cell-free synthesis reactions. These results indicate that short peptides can be effectively expressed in a cell-free translation system when the efficiency of translation was enhanced by placing a ‘booster sequence’ in front of the target peptides that are otherwise poorly expressed. The presence of the CAT-DB sequences led to the successful expression of three peptides (magainin 2, human b-defensin-2 and tachystatin A1) in sufficient amounts to be observed on a Coomassie Blue-stained gel (Fig. 3). From the relative intensity of the bands, the CAT-DB sequences corresponding to the initial 10 (CAT-DB10) to 13 (CAT-DB13) amino acids appear to be optimal for the expression of these peptides. Densitometric analysis of the bands indicated that approximately 80 (magainin 2), 450 (opistoporin), 360 (human b-defensin-2) and 120 (tachystatin A1) lg/ml of the fusion peptides were produced. Interestingly, when we introduced different silent mutations to the CAT-DB sequence, the intensity of the bands were substantially diminished (data not shown), indicating that the effect of the CAT-DB sequence addition was at least partially due to the enhanced translational efficiency by the nucleotide sequences of the CAT-DB. In order to determine if the effect of the CAT-DB sequences was due to the presence of additional nucleotide sequences or the resulting amino acid residues, indicating that the effect of the CAT-DB sequence addition was at least partially due to the enhanced translational efficiency by the nucleotide sequences of the CAT-DB. Effect of the leader sequence on the stability of cell-free synthesized peptides Short peptides are generally considered to be highly susceptible to proteolytic degradation in the cytoplasm of bacterial cells and their extracts, which was also confirmed in our experiment using chemically synthesized Ma2 as a model peptide. When Ma2 (300 lg/ml) of its authentic sequence was incubated in the same cell extract used for cell-free translation, the peptide was degraded almost completely within an hour (Fig. 4A, lane 4). However, no notable degradation was observed in the heat-treated (5 min at 80 °C) cell extract (Fig. 4A, lane 6) indicating that the endogenous proteases in the cell extract were responsible for the rapid degra-
Fig. 3. Cell-free expression of peptides with the initial codons of chloramphenicol acetyltransferase (CAT-DB) of variable lengths (number of amino acid residues from the CAT-DB are indicated).
K.-H. Lee et al. / Protein Expression and Purification 71 (2010) 16–20
19
the fusion partner, 20-fold more Ma2 (2.5 kDa) will be recovered when the same molarity of the translation product is produced. This is of particular importance in the cell-free translation systems where the synthesis of peptides is carried out using limited pool of resources including the energy sources and amino acids. In addition, since no cysteine residues are included in this fusion sequence, it would not interfere with the correct formation of disulfide bonds within the target peptides containing multiple cysteine residues. Recovery of authentic peptide molecules from in situ immobilized peptide-magnetic beads
Fig. 4. Protective effect of the CAT-DB sequences. (A) Degradation of synthetic peptide Ma2 in the cell extract. Lane 1: markers; 2: only synthetic MA2; 3: cell extracts; 4: Ma2 incubated in the cell extract for 1 h; 5: Ma2 incubated in the cell extract in the presence of protease inhibitor cocktail; 6: MA2 incubated in the heattreated cell-extracts; 7: cell-free translation of cat(10)-db ma2 during 2 h; 8: cellfree translation of cat(10)db-ma2 during 2 h and additive incubation for 1 h after the addition of chloramphenicol. (B) Degradation of native hBD2 by FXa-mediated in situ cleavage. Lane 1: markers; 2: negative control without DNA; 3: incubation of cell-free mixture with FXa; 4: cell-free translation of cat(10)db-hbd2; 5: Cell-free translation of cat(10)db-hbd2 with FXa.
dation of the peptide. Interestingly, unlike the reported results of Stephanopoulos, addition of a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) was not effective in preventing the degradation of Ma2 (Fig. 4A, lane 5). In contrast to the authentic sequence of Ma2, the translation product from the fusion construct of CAT-DB-Ma2 appeared to be highly resistant to the proteolytic digestion. After two hours of the cell-free translation reaction of cat(10)-db ma2, chloramphenicol (170 lg/ml of final concentration) was added to the reaction mixture to halt peptide synthesis. The reaction mixture was further incubated for an additional hour, and then analyzed on a Tricine– PAGE gel. As shown in Fig. 4A, the CAT-DB fused peptide maintained its intensity undeteriorated at least for 3 h. Therefore, in addition to the stimulatory effect on translation, it also appears that CAT-DB sequences improve the stability of the peptides against proteolytic digestion. The protective effect of the CAT-DB sequence does not seem to be limited to Ma2. For instance, when the hbd2 sequence fused with cat(10)-db was expressed in the cell-free translation system, the intensity of the Coomassie Blue-stained band of the translation product remained unchanged over 3 h. However, upon the removal of the auxiliary sequence (CAT-DB and hexa-histidine) by the addition of Factor Xa, instant degradation of the peptide was observed (Fig. 4B, lane 5), supporting the hypothesis that the presence of these auxiliary sequences plays an important role in protecting the peptide molecules. Taken together, we concluded that the CAT-DB sequences serve as an effective fusion partner for highyield ribosomal synthesis of peptides by both enhancing translational efficiency and protecting against proteolytic digestion. Although a number proteins have been used as the fusion partners to express peptide molecules [20], the fusion sequence designed in this study (Supplementary Table S3) offers substantial advantages compared to those conventional fusion partners. First, due to its small size (1.7–2.8 kDa), more transcriptional/translational resources can be directed for the synthesis of target peptides. For example, compared to the use of NusA (54.9 kDa) as
The open nature of cell-free translation also offers a unique advantage in the recovery of the expressed peptides. As depicted in the scheme of Fig. 1, through the expression of the designed gene constructs in the presence of affinity beads, we attempted to recover the translation product instantly after the synthesis reaction. As an example of this approach of in situ isolation, the cat-db-hexa-histidine-hbd2 construct was incubated in the reaction mixture containing Ni–NTA magnetic agarose beads. After 2 h of incubation, the magnetic beads were recovered by a magnet, washed, and then incubated in a buffer solution containing 13 lg/ml of Factor Xa for 1 h at 37 °C. When the supernatant was run on a 16% Tricine gel containing 6 M urea, a clear peptide band was observed at the expected size for human b-defensin (Fig. 5), indicating that the native peptide was successfully released from the bead leaving the auxiliary amino acid residues (CAT-DB and hexa-histidine tag) bound on the beads. It was estimated that approximately 80 lg of hBD2 was recovered from 1 ml of cell-free translation reaction mixture. Conclusion We demonstrated that the addition of properly designed leader sequences remarkably improves the productivity and convenience of peptide synthesis during cell-free translation reactions. The ability to use PCR-amplified genes as the direct template for cell-free peptide synthesis brings substantial benefits in the throughput of peptide production, compared to the conventional chemical synthesis and in vivo expression methods. Although the cell-free synthesis of peptides generally suffers from extremely low productivity, we found that the use of short CAT-DB sequence could address the issues of inefficient translation and proteolytic digestion of the expressed peptides. In addition, through the introduction of affinity tag sequence and enzyme cleavage site between the CAT-DB and target peptide sequence, target peptides of authentic amino acid sequence were easily recovered from the reaction mixture. Cost analysis of cell-free peptide synthesis indicates that the approach of cell-free synthesis can also be economically viable. As shown in Fig. 6, based on the catalogue prices of the reagents, approximately 4 USD are required for the
Fig. 5. In situ isolation of hBD2 following FXa reaction for authentic hBD2. Lane 1: markers; 2: in situ isolated CAT(10)-DB hBD2 without FXa reaction; 3: authentic hDB2 after FXa reaction.
20
K.-H. Lee et al. / Protein Expression and Purification 71 (2010) 16–20
Fig. 6. Reagent costs for a 1 ml reaction and 1 mg peptide synthesis in the cell-free system.
expression of 1 mg of hBD2. Although purification costs are not included in this estimation, considering the commercial prices of chemically synthesized peptides (approximately 100 USD for the sequence of hBD2), cell-free synthesis appears to be a feasible option at least for the generation of peptides consisted of natural amino acids. Acknowledgments Authors gratefully acknowledge financial support from the Small and Medium Business Administration of Korea (Grant No. S1033489). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.pep.2010.01.016. References [1] F. Albericio, Developments in peptide and amide synthesis, Current Opinion in Chemical Biology 8 (2004) 211–221. [2] C. Tamerler, M. Duman, E.E. Oren, M. Gungormus, X.R. Xiong, T. Kacar, B.A. Parviz, M. Sarikaya, Materials specificity and directed assembly of a goldbinding peptide, Small 2 (2006) 1372–1378.
[3] K. Rajagopal, J.P. Schneider, Self-assembling peptides and proteins for nanotechnological applications, Current Opinion in Structural Biology 14 (2004) 480–486. [4] P.W. Latham, Therapeutic peptides revisited, Nature Biotechnology 17 (1999) 755–757. [5] M. Amblard, J.A. Fehrentz, J. Martinez, G. Subra, Fundamentals of modern peptide synthesis, Methods in Molecular Biology 298 (2005) 3–24. [6] T.W. Kim, I.S. Oh, J.W. Keum, Y.C. Kwon, J.Y. Byun, K.H. Lee, C.Y. Choi, D.M. Kim, Prolonged cell-free protein synthesis using dual energy sources: combined use of creatine phosphate and glucose for the efficient supply of ATP and retarded accumulation of phosphate, Biotechnology and Bioengineering 97 (2007) 1510–1515. [7] A.R. Goerke, J.R. Swartz, High-level cell-free synthesis yields of proteins containing site-specific non-natural amino acids, Biotechnology and Bioengineering 102 (2009) 400–416. [8] T.W. Kim, J.W. Keum, I.S. Oh, C.Y. Choi, H.C. Kim, D.M. Kim, An economical and highly productive cell-free protein synthesis system utilizing fructose-1,6bisphosphate as an energy source, Journal of Biotechnology 130 (2007) 389– 393. [9] T.W. Kim, H.C. Kim, I.S. Oh, D.M. Kim, A highly efficient and economical cellfree protein synthesis system using the S12 extract of Escherichia coli, Biotechnology and Bioprocess Engineering 13 (2008) 464–469. [10] C.R. Loose, R.S. Langer, G.N. Stephanopoulos, Optimization of protein fusion partner length for maximizing in vitro translation of peptides, Biotechnology Progress 23 (2007) 444–451. [11] T.W. Kim, J.W. Keum, I.S. Oh, C.Y. Choi, C.G. Park, D.M. Kim, Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system, Journal of Biotechnology 126 (2006) 554–561. [12] M. Zasloff, Magainins, a class of antimicrobial peptides from Xenopus skin – Isolation, characterization of 2 active forms, and partial cDNA sequence of a precursor, Proceedings of the National Academy of Sciences of the United States of America 84 (1987) 5449–5453. [13] L. Moerman, S. Bosteels, W. Noppe, J. Willems, E. Clynen, L. Schoofs, K. Thevissen, J. Tytgat, J. Van Eldere, J. van der Walt, F. Verdonck, Antibacterial and antifungal properties of alpha-helical, cationic peptides in the venom of scorpions from southern Africa, European Journal of Biochemistry 269 (2002) 4799–4810. [14] D.M. Hoover, K.R. Rajashankar, R. Blumenthal, A. Puri, J.J. Oppenheim, O. Chertov, J. Lubkowski, The structure of human beta-defensin-2 shows evidence of higher order oligomerization, Journal of Biological Chemistry 275 (2000) 32911–32918. [15] N. Fujitani, S. Kawabata, T. Osaki, Y. Kumaki, M. Demura, K. Nitta, K. Kawano, Structure of the antimicrobial peptide tachystatin A, Journal of Biological Chemistry 277 (2002) 23651–23657. [16] J.M. Son, J.H. Ahn, M.Y. Hwang, C.G. Park, C.Y. Choi, D.M. Kim, Enhancing the efficiency of cell-free protein synthesis through the polymerase-chainreaction-based addition of a translation enhancer sequence and the in situ removal of the extra amino acid residues, Analytical Biochemistry 351 (2006) 187–192. [17] H. Schagger, Tricine–SDS–PAGE, Nature Protocols 1 (2006) 16–22. [18] C.M. Stenstrom, L.A. Isaksson, Influences on translation initiation and early elongation by the messenger RNA region flanking the initiation codon at the 30 side, Gene 288 (2002) 1–8. [19] J.H. Ahn, J.W. Keum, D.M. Kim, High-throughput, combinatorial engineering of initial codons for tunable expression of recombinant proteins, Journal of Proteome Research 7 (2008) 2107–2113. [20] J. Bogomolovas, B. Simon, M. Sattler, G. Stier, Screening of fusion partners for high yield expression and purification of bioactive viscotoxins, Protein Expression and Purification 64 (2009) 16–23.