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domain
Technology Research Group, The Babraham Institute, Cambridge CB2 4AT, United Kingdom
(RECEIVED July 7, 2006; FINAL REVISION September 8, 2006; ACCEPTED September 25, 2006)
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Abstract |
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 TOP Abstract IntroductionMaterials and methodsResults and DiscussionAcknowledgmentsReferences |
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light chain constant domain (C) at the C terminus of four representative proteins dramatically improved their production in the Escherichia coli S30 system, suggesting that enhancement of cell-free protein expression by C fusion will be widely applicable.
Keywords: cell-free protein expression; immunoglobulin C; domain; fusion protein
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Introduction |
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TOPAbstractIntroductionMaterials and methodsResults and DiscussionAcknowledgmentsReferences |
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Several established systems are available, including rabbit reticulocyte, Escherichia coli S30, and wheat germ lysates, and more recently, mammalian cell extracts (Mikami et al. 2006) and the artificially assembled PURE system (Shimizu et al. 2001) have been introduced. Efforts have been made to improve protein yield by identifying key factors affecting in vitro transcription and translation and developing modified protocols (Sawasaki et al. 2002; Spirin 2004; Calhoun and Swartz 2005). They include the composition of the system itself, e.g., extracts of genetically engineered bacterial strains, various energy resources or amino acid concentrations, or use of defined components. Second, various production conditions have been used, such as dialysis, continuous flow, continuous exchange, hollow fiber, and bilayer systems (Sawasaki et al. 2002; Calhoun and Swartz 2005). Despite these developments, some proteins are still only poorly expressed (or not at all) in cell-free systems. Codon optimization can be useful, but is time-consuming and often requires the assistance of prediction software.
Fusion of proteins to additional domains is widely used as a means of improving solubility and stability in heterologous in vivo expression systems (Shaki-Loewenstein et al. 2005). Popular tags include maltose-binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX), and NusA. Recently, fusion to a well-expressed N-terminal sequence of chloramphenicol acetyl transferase (CAT) has been reported to increase protein expression by up to 14-fold in an E. coli lysate (Son et al. 2006). The constant domain of the immunoglobulin light chain (C) has been used as a C-terminal fusion with single chain antibody fragments (scAb) and T-cell receptors (TCRs) to improve E. coli expression in vivo (Maynard et al. 2002, 2005) and as a spacer for scAb during ribosome display in vitro (He and Taussig 1997). However, it has not been applied so far to other proteins or for in vitro expression. Here, we report that fusion of the human C domain at the C terminus of several poorly expressed proteins significantly improves their expression in the E. coli S30 system. The use of C fusions thus provides a new approach to enhanced cell-free protein production. Moreover, the C domain can be used for immunodetection and affinity purification.
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Materials and methods |
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TOPAbstractIntroductionMaterials and methodsResults and DiscussionAcknowledgmentsReferences |
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Molecular biology reagents and cell-free system
Nucleotides, agarose, the PCR Gel Extraction Kit, and the HRP-linked mouse anti-His antibody were from Sigma; Taq DNA polymerase from QIAGEN; HRP-linked anti-human antibody from The Binding Site; NuPAGE Bis-Tris gels from Invitrogen; PVDF Immobilon-P membranes from Millipore; Western Blot detection SuperSignal Kit from Pierce; and the coupled E. coli S30 cell-free expression system from Roche.
Construction of PCR fragments
The general PCR constructs used for cell-free protein synthesis are shown in Figure 1A. The 5'-end contains a T7 promoter, a gene 10 enhancer, and an SD sequence (Roche kit) for efficient transcription and translation. The open reading frame (ORF) of the gene of interest was placed after the initiation codon ATG, followed by fusion in frame to the following in order: a flexible peptide linker, a double-(His)6 tag, and two consecutive stop codons (TAATAA) (He and Taussig 2001). When human C was included, it was placed between the gene ORF and the peptide linker. A transcription termination region was included at the 3'-end of the constructs.
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-d(His)6 tag domain was produced using primers HuC4/B and Tterm/F on a plasmid template, which encodes the C domain with the double-(His)6 tag fused at the C terminus. , primers PErzpd/B and Rzpd–L/F were used, while PErzpd/B and RzpdCk/F were used for constructs with C.
Assembly PCR
The ORF of the gene of interest and the appropriate domain fragments were assembled by mixing in equimolar ratios (total DNA 50–100 ng) after elution from agarose gel (1%); adding into a PCR solution containing 2.5 µL of 10x PCR buffer, 1 µL of dNTP mix containing 2.5 mM each, 1 U of Taq DNA polymerase, and water to a final volume of 25 µL; and thermal cycling for eight cycles (94°C for 30 sec, 54°C for 1 min, and 72°C for 1 min). For constructs without C, the fragments assembled were the RTST7 domain, gene ORF, and the double-(His)6 tag domain, while for the constructs with C, they were the RTST7 domain, gene ORF, and the C-d(His)6 tag domain.
Amplification of PCR constructs
Assembled constructs were amplified by transferring 2 µL to a second PCR mixture in a final volume of 50 µL (as above) for a further 30 cycles using primers RTST7/B and T-term/F. Thermal cycling for 30 cycles (94°C for 30 sec, 54°C for 1 min, and 72°C for 1 min; finally, 72°C for 8 min). The final PCR construct was analyzed by agarose (1%) gel electrophoresis to determine quality and concentration by comparison with a known DNA marker. The PCR products may be used for cell-free expression with or without further purification.
Cell-free protein synthesis
Proteins were expressed from PCR constructs using the coupled E. coli S30 system, incubated for 4 h at 30°C. A standard reaction comprised 12 µL of E. coli S30 lysate, 12 µL of amino acids, 10 µL of reaction mix, 5 µL of reconstitution buffer, 1 µL of methionine, and 100–500 ng of PCR DNA, made to 50 µL with water. For a small-scale expression, 5–10 µL of the total reaction mixture was used.
Detection of proteins by Western blotting
Proteins expressed in the E. coli S30 lysate were mixed with an equal volume of 2x SDS buffer (100 mM Tris at pH 8.0, 5% SDS, 0.2% bromophenol blue, 20% glycerol), heated to 90°C for 5 min, loaded onto a 10% NuPAGE Bis-Tris gel, and run at 200 V. The separated bands were transferred to a PVDF membrane by electroblotting for 2 h at 80 mA. The membrane was blocked in 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 h, then incubated with either HRP-linked mouse anti-His antibody (diluted 1:4000 in PBS/BSA) or HRP-linked mouse anti- antibody (1:500 in PBS/BSA) for 1 h. The membrane was developed using the SuperSignal kit (Pierce) as per the manufacturer's instructions.
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Results and Discussion |
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TOPAbstractIntroductionMaterials and methodsResults and DiscussionAcknowledgmentsReferences |
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domain. The two-domain (VH, VL) scFv construct is a standard format for cell-based recombinant antibody expression (Holliger and Hudson 2005). Anti-carcinoembryonic antigen (CEA) and anti-progesterone scFv fragments, created previously by ribosome display (He and Taussig 1997), were assembled as fusions to a double-hexahistidine tag sequence [double-(His)6] (He and Taussig 2001) or additionally to the C domain [C-double-(His)6] by PCR (Fig. 1A; Materials and Methods). After expression in the E. coli S30 system, Western blotting and detection by monoclonal anti-His antibody showed that neither scFv-double-(His)6 fragment was expressed detectably, whereas both scFv-C-double-(His)6 fusions successfully led to high expression yields (Fig. 1B,C). Comparison with protein standards estimated that at least 100 µg/mL protein was produced after inclusion of the C domain. Sequencing of the PCR constructs confirmed that the reading frames were in all cases correct and that the only sequence differences were the presence or absence of C. It was also shown that the scFv-C-double-(His)6 fragments were retained in the soluble fraction after high-speed centrifugation (15,000 rpm for 20 min) and bound their respective specific antigens (data not shown).
To test whether C-terminal fusion to the C domain could also improve expression of other proteins known to be synthesized at very low levels, we selected Rab22b (a GTP binding protein) and FKBP2 (FK506 binding protein). As double-(His)6 constructs, both were barely detectable using anti-His antibody after expression in the E. coli S30 system (Fig. 1D,F, tracks 1). In contrast, the yields of Rab22b-C-double-(His)6 and FKBP2-C-double-(His)6 were high, and they were strongly detected on Western blotting by anti-His (Fig. 1D,F, tracks 2) and anti-C monoclonal antibodies (Fig. 1E,G). Where the non-C-tagged protein was detectable on a Western blot, the increased expression through inclusion of the C domain was estimated as at least 10–50-fold.
Alternative tags, such as the CAT sequence, have been successfully added to target genes to increase expression, but have in general been N-terminal fusions (Shaki-Loewenstein et al. 2005; Son et al. 2006). Well-expressed N-terminal tags increase translation initiation and thus production of the overall protein. In contrast, our work involves the novel use of a tag at the C terminus. Reasons for the increased expression are speculative, and effects on transcription or mRNA structure and stability cannot be excluded. Others have reported that addition of C to antibody fragments increases protein expression and thermal stability in vivo (Hayhurst 2000; Maynard et al. 2002), and cellular expression of certain recombinant TCRs in E. coli is also improved by C fusion (Maynard et al. 2005). It is possible that C stabilizes nascent polypeptides during cell-free synthesis, improves folding, or protects the fused proteins from degradation. However, in this study, all the constructs contain identical N and C termini (Fig. 1A), so that the increased expression of the C-tagged proteins is unlikely to be explained by reduced C-terminal proteolysis.
In conclusion, our results demonstrate that fusion to the human C domain leads to high level expression of otherwise scarcely or very weakly expressed proteins in a standard cell-free system. We have shown that this is not restricted to antibody fragments, but can also be applied to unrelated, non-Ig domain proteins. Moreover, the availability of detection and binding reagents (e.g., antibodies, protein L) means that the C domain can be used both in Western blotting and other immunoassays, as well as in affinity purification, making it potentially useful as a multipurpose fusion tag. It has also been shown that a tag can be removed from an expressed protein by in situ specific cleavage at an engineered protease site in an E. coli cell-free translation mixture (Son et al. 2006), which could be applied to generate the non-fusion protein as required. Recently, cell-free synthesized proteins have been directly used for studies by mass spectrometry or NMR (Jungbauer and Cavagnero 2006; Keppetipola et al. 2006). Thus, C-domain fusions could find wide applicability in protein expression for structural and functional studies.
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Footnotes |
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Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062429906.
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Acknowledgments |
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TOPAbstractIntroductionMaterials and methodsResults and DiscussionAcknowledgmentsReferences |
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References |
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TOPAbstractIntroductionMaterials and methodsResults and DiscussionAcknowledgmentsReferences |
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Hanes, J. and Plückthun, A. 1997. In vitro selection and evolution of functional proteins by using ribosome display. Proc. Natl. Acad. Sci. 94: 4937–4942.
Hayhurst, A. 2000. Improved expression characteristics of single-chain Fv fragments when fused downstream of the E. coli maltose-binding protein or upstream of a single immunoglobulin-constant domain. Protein Expr. Purif. 18: 1–10.[CrossRef][Medline]
He, M. and Taussig, M.J. 1997. Antibody–ribosome–mRNA (ARM) complexes as efficient selection particles for in vitro display and evolution of antibody combining sites. Nucleic Acids Res. 25: 5132–5134.
He, M. and Taussig, M.J. 2001. Single step generation of protein arrays from DNA by cell-free expression and in situ immobilization (PISA method). Nucleic Acids Res. 29: e73.
Holliger, P. and Hudson, P.J. 2005. Engineering antibody fragments and the rise of single domains. Nat. Biotechnol. 23: 1126–1136.[CrossRef][Medline]
Jungbauer, L.M. and Cavagnero, S. 2006. Characterization of protein expression and folding in cell-free system by MALDI-TOF mass spectrometry. Anal. Chem. 78: 2841–2852.[Medline]
Keppetipola, S., Kudlicki, W., Nguyen, B.D., Meng, X., Donovan, K.J., and Shaka, A.J. 2006. From gene to HSQC in under five hours: High-throughput NMR proteomics. J. Am. Chem. Soc. 128: 4508–4509.[CrossRef][Medline]
Maynard, J.A., Maassen, C.B., Leppla, S.H., Brasky, K., Patterson, J.L., Iverson, B.L., and Georgiou, G. 2002. Protection against anthrax toxin by recombinant antibody fragments correlates with antigen affinity. Nat. Biotechnol. 20: 597–601.[CrossRef][Medline]
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Sawasaki, T., Hasegawa, Y., Tsuchimochi, M., Kamura, N., Ogasawara, T., Kuroita, T., and Endo, Y. 2002. A bilayer cell-free protein synthesis system for high-throughput screening of gene products. FEBS Lett. 514: 102–105.[CrossRef][Medline]
Shaki-Loewenstein, S., Zfania, R., Hyland, S., Wels, W.S., and Benhar, I. 2005. A universal strategy for stable intracellular antibodies. J. Immunol. Method. 303: 19–39.[CrossRef][Medline]
Shimizu, Y., Inoue, A., Tomari, Y., Suzuki, T., Yokogawa, T., Nishikawa, K., and Ueda, T. 2001. Cell-free translation reconstituted with purified components. Nat. Biotechnol. 19: 751–755.[CrossRef][Medline]
Son, J.M., Ahn, J.H., Hwang, M.Y., Park, C.G., Choi, C.Y., and Kim, D.M. 2006. Enhancing the efficiency of cell-free protein synthesis through the polymerase-chain-reaction-based addition of a translation enhancer sequence and the in situ removal of the extra amino acid residues. Anal. Biochem. 351: 187–192.[CrossRef][Medline]
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