enhanced cell-free protein expression by fusion with immunoglobulin cκ domain

FOR THE RECORD

Enhanced cell-free protein expression by fusion withimmunoglobulin Ck domain

ELIZABETH PALMER, HONG LIU, FARID KHAN, MICHAEL J. TAUSSIG,AND MINGYUE HETechnology Research Group, The Babraham Institute, Cambridge CB2 4AT, United Kingdom

(RECEIVED July 7, 2006; FINAL REVISION September 8, 2006; ACCEPTED September 25, 2006)

Abstract

While cell-free systems are increasingly used for protein expression in structural and functional studies,several proteins are difficult to express or expressed only at low levels in cell-free lysates. Here, wereport that fusion of the human immunoglobulin k light chain constant domain (Ck) at the C terminus offour representative proteins dramatically improved their production in the Escherichia coli S30 system,suggesting that enhancement of cell-free protein expression by Ck fusion will be widely applicable.

Keywords: cell-free protein expression; immunoglobulin Ck; domain; fusion protein

Production of proteins in heterologous systems is a majorchallenge in many areas of biological research and bio-pharmaceutical development. Cell-free protein synthesisis becoming a widely used alternative to cell-based meth-ods for parallel production of proteins, providing a rapidroute to the translation of genetic information into func-tional proteins (Spirin 2004). Like in vitro methods, cell-free expression systems also allow proteins to be expressedand modified during translation under defined conditionsthat living cells may be incapable of reproducing. Severalsignificant protein selection and display technologies, in-cluding ribosome display, mRNA display, and in situ proteinarrays, also make use of cell-free protein expression systems(Hanes and Pluckthun 1997; He and Taussig 1997, 2001).

Several established systems are available, includingrabbit reticulocyte, Escherichia coli S30, and wheat germlysates, and more recently, mammalian cell extracts(Mikami et al. 2006) and the artificially assembled PUREsystem (Shimizu et al. 2001) have been introduced.

Efforts have been made to improve protein yield byidentifying key factors affecting in vitro transcriptionand translation and developing modified protocols(Sawasaki et al. 2002; Spirin 2004; Calhoun and Swartz2005). They include the composition of the system itself,e.g., extracts of genetically engineered bacterial strains,various energy resources or amino acid concentrations, oruse of defined components. Second, various productionconditions have been used, such as dialysis, continuousflow, continuous exchange, hollow fiber, and bilayer systems(Sawasaki et al. 2002; Calhoun and Swartz 2005). Despitethese developments, some proteins are still only poorlyexpressed (or not at all) in cell-free systems. Codon op-timization can be useful, but is time-consuming and oftenrequires the assistance of prediction software.

Fusion of proteins to additional domains is widely usedas a means of improving solubility and stability in heter-ologous in vivo expression systems (Shaki-Loewensteinet al. 2005). Popular tags include maltose-binding protein(MBP), glutathione S-transferase (GST), thioredoxin (TRX),and NusA. Recently, fusion to a well-expressed N-terminalsequence of chloramphenicol acetyl transferase (CAT) hasbeen reported to increase protein expression by up to 14-fold in an E. coli lysate (Son et al. 2006). The constantdomain of the immunoglobulin k light chain (Ck) has beenused as a C-terminal fusion with single chain antibodyfragments (scAb) and T-cell receptors (TCRs) to improve

ps0624299 Palmer et al. FOR THE RECORD RA

Reprint requests to: Michael J. Taussig, Technology ResearchGroup, The Babraham Institute, Cambridge CB2 4AT, UK; e-mail:[email protected]; fax: 44-1223-496045; or Mingyue He,Technology Research Group, The Babraham Institute, CambridgeCB2 4AT, UK; e-mail: [email protected]; fax: 44-1223-496045.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062429906.

2842 Protein Science (2006), 15:2842–2846. Published by Cold Spring Harbor Laboratory Press. Copyright � 2006 The Protein Society

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E. coli expression in vivo (Maynard et al. 2002, 2005) andas a spacer for scAb during ribosome display in vitro (Heand Taussig 1997). However, it has not been applied so farto other proteins or for in vitro expression. Here, we reportthat fusion of the human Ck domain at the C terminus ofseveral poorly expressed proteins significantly improvestheir expression in the E. coli S30 system. The use of Ckfusions thus provides a new approach to enhanced cell-freeprotein production. Moreover, the Ck domain can be usedfor immunodetection and affinity purification.

Materials and methods

Primers

The primers used in this study are as follows:

RTST7/B: 59-GATCTCGATCCCGCG-39PET7/F: 59-CATGGTGGATATCTCCTTCTTAAAG-39Linker-tag/B: 59-GCTCTAGAGGCGGTGGC-39Tterm/F: 59-TCCGGATATAGTTCCTCC-39HuC4/B: 59-GTGGCTGCACCATCTGTCT-39RzpdCk/F: 59-AGATGGTGCAGCCACAGTTTTGTACAAGAAAGCTGGG-39

PErzpd/B: 59-CTTAAGAAGGAGATATCCACCATGCTCGAATCAACAAGTTTGTAC-39

Rzpd–L/F: 59-GCCACCGCCTCTAGAGCGTTTGTACAAGAAAGCTGG-39

Molecular biology reagents and cell-free system

Nucleotides, agarose, the PCR Gel Extraction Kit, and theHRP-linked mouse anti-His antibody were from Sigma;Taq DNA polymerase from QIAGEN; HRP-linked anti-human k antibody from The Binding Site; NuPAGE Bis-Tris gels from Invitrogen; PVDF Immobilon-P mem-branes from Millipore; Western Blot detection Super-Signal 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 proteinsynthesis are shown in Figure 1A. The 59-end containsa T7 promoter, a gene 10 enhancer, and an SD sequence(Roche kit) for efficient transcription and translation. Theopen reading frame (ORF) of the gene of interest wasplaced after the initiation codon ATG, followed by fusionin frame to the following in order: a flexible peptidelinker, a double-(His)6 tag, and two consecutive stopcodons (TAATAA) (He and Taussig 2001). When humanCk was included, it was placed between the gene ORFand the peptide linker. A transcription termination regionwas included at the 39-end of the constructs.

PCR generation of individual domainsThe standard PCR mixture consisted of 5 mL of 103

PCR buffer, 10 mL of 53 Q, 4 mL of dNTP mixcontaining 2.5 mM each, 1.5 mL of forward and backwardprimers (16 mM each), 1 U of Taq DNA polymerase, 1–10ng of template DNA, and water to a final volume of 50mL.

(1) RTST7 domain, comprising T7 promoter, gene 10enhancer, and SD sequence, was created using pri-mers RTST7/B and PET7/F from a plasmid templateused as a control in the cell-free system (Roche).

(2) Double-(His)6 tag domain, comprising a flexible pep-tide linker, two hexahistidine sequences, separated byan 11-amino-acid spacer sequence, and two consecu-tive stop codons (TAATAA), was generated usingprimers Linker-tag/B and Tterm/F on the plasmidtemplate pTA-His (He and Taussig 2001).

(3) Ck-d(His)6 tag domain was produced using primersHuC4/B and Tterm/F on a plasmid template, whichencodes the Ck domain with the double-(His)6 tagfused at the C terminus.

(4) Open reading frame (ORF) of genes to be expressedwas amplified using their corresponding plasmids(RZPD German Genome Resource Center, Heidel-berg) as templates and individually designed primers.For generation of constructs without Ck, primersPErzpd/B and Rzpd–L/F were used, while PErzpd/Band RzpdCk/F were used for constructs with Ck.

Assembly PCRThe ORF of the gene of interest and the appropriate

domain fragments were assembled by mixing in equimo-lar ratios (total DNA 50–100 ng) after elution fromagarose gel (1%); adding into a PCR solution containing2.5 mL of 103 PCR buffer, 1 mL of dNTP mix containing2.5 mM each, 1 U of Taq DNA polymerase, and water toa final volume of 25 mL; and thermal cycling for eightcycles (94°C for 30 sec, 54°C for 1 min, and 72°C for 1min). For constructs without Ck, the fragments assembledwere the RTST7 domain, gene ORF, and the double-(His)6tag domain, while for the constructs with Ck, they were theRTST7 domain, gene ORF, and the Ck-d(His)6 tag domain.

Amplification of PCR constructsAssembled constructs were amplified by transferring 2

mL to a second PCR mixture in a final volume of 50 mL(as above) for a further 30 cycles using primers RTST7/Band T-term/F. Thermal cycling for 30 cycles (94°C for 30sec, 54°C for 1 min, and 72°C for 1 min; finally, 72°C for8 min). The final PCR construct was analyzed by agarose(1%) gel electrophoresis to determine quality and con-centration by comparison with a known DNA marker. The

Cell-free expression of Ck fusion proteins

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PCR products may be used for cell-free expression withor without further purification.

Cell-free protein synthesis

Proteins were expressed from PCR constructs using thecoupled E. coli S30 system, incubated for 4 h at 30°C. Astandard reaction comprised 12 mL of E. coli S30 lysate, 12mL of amino acids, 10 mL of reaction mix, 5 mL ofreconstitution buffer, 1 mL of methionine, and 100–500 ngof PCR DNA, made to 50 mL with water. For a small-scaleexpression, 5–10 mL of the total reaction mixture was used.

Detection of proteins by Western blotting

Proteins expressed in the E. coli S30 lysate were mixedwith an equal volume of 23 SDS buffer (100 mM Tris atpH 8.0, 5% SDS, 0.2% bromophenol blue, 20% glycerol),heated to 90°C for 5 min, loaded onto a 10% NuPAGEBis-Tris gel, and run at 200 V. The separated bands weretransferred to a PVDF membrane by electroblotting for 2h at 80 mA. The membrane was blocked in 1% bovineserum albumin (BSA) in phosphate-buffered saline (PBS)for 1 h, then incubated with either HRP-linked mouseanti-His antibody (diluted 1:4000 in PBS/BSA) or HRP-linked mouse anti-k antibody (1:500 in PBS/BSA) for 1 h.

Figure 1. Cell-free expression of proteins with and without Ck domain fusion. (A) Structures of constructs without (i) and with (ii) the

Ck domain. (RTST7) T7 promoter, gene 10 enhancer and Shine-Delgarno sequence; (ORF) open reading frame; [Double (His)6] two

hexahistidines separated by an 11-amino-acid spacer, joined to the protein via a flexible linker sequence; (Termination sequence)

two consecutive stop codons (TAATAA). (B–F) Western blotting of expressed proteins. [d(His)6] Double-(His)6 tag. (B) Anti-CEA

antibody 636 scFv without (track 1) or with Ck domain (track 2), detected by anti-His antibody. (C) Anti-progesterone antibody 941

scFv without (track 1) or with Ck domain (track 2), detected by anti-His antibody. (D) Rab22b without (track 1) or with the Ck domain

(track 2), detected by anti-His antibody. (E) Rab22b without (track 1) or with the Ck domain (track 2), detected by anti-Ck antibody.

(F) FKBP2 without (track 1) or with the Ck domain (track 2), detected by anti-His antibody. (G) FKBP2 without (track 1) or with the

Ck domain (track 2), detected by anti-Ck antibody.

Palmer et al.

2844 Protein Science, vol. 15



The membrane was developed using the SuperSignal kit(Pierce) as per the manufacturer’s instructions.

Results and Discussion

As a first example, we compared expression of humanscAb fragment constructs with and without the humanCk domain. The two-domain (VH, VL) scFv construct isa standard format for cell-based recombinant antibodyexpression (Holliger and Hudson 2005). Anti-carcinoem-bryonic antigen (CEA) and anti-progesterone scFv frag-ments, created previously by ribosome display (He andTaussig 1997), were assembled as fusions to a double-hexahistidine tag sequence [double-(His)6] (He andTaussig 2001) or additionally to the Ck domain [Ck-double-(His)6] by PCR (Fig. 1A; Materials and Methods).After expression in the E. coli S30 system, Westernblotting and detection by monoclonal anti-His antibodyshowed that neither scFv-double-(His)6 fragment wasexpressed detectably, whereas both scFv-Ck-double-(His)6 fusions successfully led to high expression yields(Fig. 1B,C). Comparison with protein standards estimatedthat at least 100 mg/mL protein was produced afterinclusion of the Ck domain. Sequencing of the PCRconstructs confirmed that the reading frames were in allcases correct and that the only sequence differences werethe presence or absence of Ck. It was also shown that thescFv-Ck-double-(His)6 fragments were retained in thesoluble fraction after high-speed centrifugation (15,000rpm for 20 min) and bound their respective specific antigens(data not shown).To test whether C-terminal fusion to the Ck domain

could also improve expression of other proteins known tobe synthesized at very low levels, we selected Rab22b (aGTP binding protein) and FKBP2 (FK506 binding pro-tein). As double-(His)6 constructs, both were barelydetectable using anti-His antibody after expression inthe E. coli S30 system (Fig. 1D,F, tracks 1). In contrast,the yields of Rab22b-Ck-double-(His)6 and FKBP2-Ck-double-(His)6 were high, and they were strongly detectedon Western blotting by anti-His (Fig. 1D,F, tracks 2) andanti-Ck monoclonal antibodies (Fig. 1E,G). Where thenon-Ck-tagged protein was detectable on a Western blot,the increased expression through inclusion of the Ckdomain 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-expressedN-terminal tags increase translation initiation and thusproduction of the overall protein. In contrast, our workinvolves the novel use of a tag at the C terminus. Reasonsfor the increased expression are speculative, and effectson transcription or mRNA structure and stability cannot

be excluded. Others have reported that addition of Ck toantibody fragments increases protein expression and ther-mal stability in vivo (Hayhurst 2000; Maynard et al. 2002),and cellular expression of certain recombinant TCRs inE. coli is also improved by Ck fusion (Maynard et al. 2005).It is possible that Ck stabilizes nascent polypeptides duringcell-free synthesis, improves folding, or protects the fusedproteins from degradation. However, in this study, all theconstructs contain identical N and C termini (Fig. 1A), sothat the increased expression of the Ck-tagged proteins isunlikely to be explained by reduced C-terminal proteolysis.

In conclusion, our results demonstrate that fusion tothe human Ck domain leads to high level expression ofotherwise scarcely or very weakly expressed proteins ina standard cell-free system. We have shown that this isnot restricted to antibody fragments, but can also beapplied to unrelated, non-Ig domain proteins. Moreover,the availability of detection and binding reagents (e.g.,antibodies, protein L) means that the Ck domain can beused both in Western blotting and other immunoassays, aswell as in affinity purification, making it potentially useful asa multipurpose fusion tag. It has also been shown that a tagcan be removed from an expressed protein by in situ specificcleavage at an engineered protease site in an E. coli cell-freetranslation mixture (Son et al. 2006), which could be appliedto generate the non-fusion protein as required. Recently, cell-free synthesized proteins have been directly used for studiesby mass spectrometry or NMR (Jungbauer and Cavagnero2006; Keppetipola et al. 2006). Thus, Ck-domain fusionscould find wide applicability in protein expression for struc-tural and functional studies.

Acknowledgments

We thank Bernhard Korn (Heidelberg) for providing the Rab22band FKBP2 clones. This work was supported through the EC6th Framework Programme Integrated Project MolTools. Workat the Babraham Institute is supported by the BBSRC, UK.

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enhanced cell-free protein expression by fusion with immunoglobulin cκ domain

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