A new method to customize protein expression vectors for fast, efficient and background free parallel cloning

BMC Biotechnology

Judith Scholz¹  Hüseyin Besir²  Claudia Strasser¹  Sabine Suppmann¹ 

[1] Max-Planck Institute of Biochemistry, Am Klopferspitz 18, 82152, Martinsried, Germany

[2] EMBL Heidelberg, Meyerhofstrasse 1, 69117, Heidelberg, Germany

关键词: Protein production;    Mammalian expression;    Insect cell expression;    Bacterial expression;    Expression screening;    Restriction enzyme free cloning;    Parallel cloning;   

Others  :  1123264
DOI  :  10.1186/1472-6750-13-12

 received in 2012-08-21, accepted in 2012-12-21,  发布年份 2013

【 摘 要 】

Background

Expression and purification of correctly folded proteins typically require screening of different parameters such as protein variants, solubility enhancing tags or expression hosts. Parallel vector series that cover all variations are available, but not without compromise. We have established a fast, efficient and absolutely background free cloning approach that can be applied to any selected vector.

Results

Here we describe a method to tailor selected expression vectors for parallel Sequence and Ligation Independent Cloning. SLIC cloning enables precise and sequence independent engineering and is based on joining vector and insert with 15–25 bp homologies on both DNA ends by homologous recombination. We modified expression vectors based on pET, pFastBac and pTT backbones for parallel PCR-based cloning and screening in E.coli, insect cells and HEK293E cells, respectively. We introduced the toxic ccdB gene under control of a strong constitutive promoter for counterselection of insert less vector. In contrast to DpnI treatment commonly used to reduce vector background, ccdB used in our vector series is 100% efficient in killing parental vector carrying cells and reduces vector background to zero. In addition, the 3’ end of ccdB functions as a primer binding site common to all vectors. The second shared primer binding site is provided by a HRV 3C protease cleavage site located downstream of purification and solubility enhancing tags for tag removal. We have so far generated more than 30 different parallel expression vectors, and successfully cloned and expressed more than 250 genes with this vector series. There is no size restriction for gene insertion, clone efficiency is > 95% with clone numbers up to 200. The procedure is simple, fast, efficient and cost-effective. All expression vectors showed efficient expression of eGFP and different target proteins requested to be produced and purified at our Core Facility services.

Conclusion

This new expression vector series allows efficient and cost-effective parallel cloning and thus screening of different protein constructs, tags and expression hosts.

【 授权许可】

   
2013 Scholz et al; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Vincentelli R, Cimino A, Geerlof A, Kubo A, Satou Y, Cambillau C: High-throughput protein expression screening and purification in Escherichia coli. Methods 2011, 55(1):65-72.
• [2]An Y, Yumerefendi H, Mas PJ, Chesneau A, Hart DJ: ORF-selector ESPRIT: a second generation library screen for soluble protein expression employing precise open reading frame selection. J Struct Biol 2011, 175(2):189-197.
• [3]Cabantous S, Waldo GS: In vivo and in vitro protein solubility assays using split GFP. Nat Methods 2006, 3(10):845-854.
• [4]Engler C, Gruetzner R, Kandzia R, Marillonnet S: Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes. PlosOne 2009, 4(5):e5553.
• [5]Unger T, Jacobovitch Y, Dantes A, Bernheim R, Peleg Y: Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression. J Struct Biol 2010, 172(1):34-44.
• [6]Li MZ, Elledge SJ: Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Methods 2007, 4(3):251-256.
• [7]Berrow NS, Alderton D, Sainsbury S, Nettleship J, Assenberg R, Rahman N, Stuart DI, Owens RJ: A versatile ligation-independent cloning method suitable for high-throughput expression screening applications. Nucleic Acids Res 2007, 35(6):e45.
• [8]Dümmler A, Lawrence AM, de Marco A: Simplified screening for the detection of soluble fusion constructs expressed in E. coli using a modular set of vectors. Microb Cell Fact 2005, 4:34. BioMed Central Full Text
• [9]Lancini G, Parenti F: Antibiotics. New York: Springer; 1982.
• [10]Geisse S: Reflections on more than 10 years of TGE approaches. Protein Expr Purif 2009, 64:99-107.
• [11]Durocher Y, Perret S, Kamen A: High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res 2002, 30(2):E9.
• [12]Carr S, Miller J, Leary SE, Bennett AM, Ho A, Williamson ED: Expression of a recombinant form of the V antigen of Yersinia pestis, using three different expression systems. Vaccine 1999, 18:153-159.
• [13]Bernard P, Gabant P, Bahassi EM, Couturier M: Positive-selection vectors using the F plasmid ccdB killer gene. Gene 1994, 148(1):71-74.
• [14]Inouye S, Inouye M: Up-promoter mutations in the lpp gene of Escherichia coli. Nucl. Acids Res 1985, 13(9):3101-3110.
• [15]Reverter D, Lima CD: A basis for SUMO protease specificity provided by analysis of human Senp2 and a Senp2-SUMO complex. Structure 2004, 12(8):1519-1531.
• [16]Breitsprecher D, Kiesewetter AK, Linkner J, Faix J: Analysis of actin assembly by in vitro TIRF microscopy. Methods Mol Biol 2009, 571:401-415.
• [17]Chen Z, Yang H, Pavletich NP: Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structures. Nature 2008, 453(7194):489-4.
• [18]Kilunc MO, Mukundan L, Yolcu ES, Singh NP, Suttles J, Shirwan H: Generation of a multimeric form of CD40L with potent immunostimulatory activity using streptavidin as a chaperon. Exp Mol Pathol 2006, 80:252-261.
• [19]Shen A, Lupardu PJ, Morell M, Ponder EL, Sadaghiani AM, Garcia KC Bogyo M: Simplified, Enhanced Protein Purification Using an Inducible, Autoprocessing Enzyme Tag. PlosOne 2009, 4(12):e8119.
• [20]Li MZ, Elledge SJ: SLIC a method for sequence- and ligation-independent cloning. Methods Mol Biol 2012, 852:51-59.
• [21]Studier FW: Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 2005, 41(1):207-234.
• [22]Hopkins R, Esposito D: A rapid method for titrating baculovirus stocks using the Sf-9 Easy Titer cell line. Biotechniques 2009, 47(3):785-788.
• [23]Wasilko DJ, Lee SE: TIPS: titerless infected-cells preservation and scale-up. Bioprocess J. 2006, 5:29-32.
• [24]Tom R, Bisson L, Durocher Y: Transient expression in HEK293-EBNA1 cells. In Expression systems. Edited by Dyson MR, Durocher Y. UK: Scion Publishing Ltd; 2007:212-218.