Springer Protocols: Abstract: SIMPLEX: Single-Molecule PCR-Linked In Vitro Expression: A Novel Method for High-Throughput Construction and Screening of Protein Libraries

SIMPLEX: Single-Molecule PCR-Linked In Vitro Expression: A Novel Method for High-Throughput Construction and Screening of Protein Libraries

By: Suang Rungpragayphan², Tsuneo Yamane³, Hideo Nakano⁴

Abstract

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A novel strategy for construction of protein libraries called “SIMPLEX: single-molecule PCR-linked in vitro expression” is described. A pool of genes is prepared and thereafter extensively diluted to give one molecule of DNA per well. Each individual molecule is separately amplified by PCR (single-molecule PCR) yielding a one-well-one-gene PCR library. Subsequently, the PCR library is directly transformed into a protein library by means of in vitro-coupled transcription/translation in an array format. Individual proteins in the library can be screened for target functions directly without further purification. The generated protein library is compatible with various selection methods. The strategy provides high-throughput construction and screening of protein libraries, and suits automation.

Affiliation(s): (2) Department of Biopharmacy, Faculty of Pharmacy, Silpakorn University, Nakorn-Pathom, Thailand
(3) Department of Environmental Biology, College of Bioscience and Biotechnology, Chubu University, Kasugai, Japan
(4) Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan

Book Title: In Vitro Transcription and Translation Protocols

Series: Methods in Molecular Biology | Volume: 375 | Pub. Date: May-03-2007 | Page Range: 79-94 | DOI: 10.1007/978-1-59745-388-2_4

Subject: Genetics/Genomics

Key Words: SIMPLEX - single-molecule PCR - in vitro expression - coupled transcription/translation - PCR library - protein library - protein engineering - high-throughput screening

References

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1.	Mao, S., Gao, C., Lo, C. L., Wirshing, P., Wong, C., and Janda, K. D. (1999) Phage-display library selection of high-affinity human single-chain antibodies to tumor-associated carbohydrate antigens sialyl Lewisx and Lewisx. Proc. Natl. Acad. Sci. USA 96, 6953–6958.

2.	Zavala, A. G., Lancaster, T., Groopman, J. D., Stickland, P. T., and Chandrasegaran, S. (2000) Phage display of ScFv peptides recognizing the thymidine(6-4)thymidine photoproduct. Nucleic Acids Res. 28, e24.

3.	Rodi, D.J. and Makowski, L. (1999) Phage-display technology-finding a needle in a vast molecular haystack. Curr. Opin. Biotechnol. 10, 87–93.

4.	Cull, M. G., Miller, J.F., and Schatz, P. J. (1992) Screening for receptor ligands using large libraries of peptides linked to the C terminus of the lac repressor. Proc. Natl. Acad. Sci. USA 89, 1865–1869.

5.	Georgiou, G., Stathopoulos, C., Daugherty, P. S., Nayak, A. R., Iverson, B. L. and Ill, R. C. (1996) Display of heterologous proteins on the surface of microorganisms: from the screening to live recombinant vaccines. Nat. Biotechnol. 15, 29–33.

6.	Boder, E.T. and Wittrup, K. D. (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat. Biotechnol. 15, 553–557.

7.	eke, M. C., Cho, B. K., Boder, E. T., Kranz, D. M., and Wittrup, K. D. (1997) Isolation of anti-T cell receptor scFv mutants by yeast surface display. Protein Eng. 10, 1303–1310.

8.	Cho, B. K., Kieke, M. C., Boder, E. T., Wittrup, K. D., and Kranz, D. M. (1998) A yeast surface display system for the discovery of ligands that trigger cell activation. J. Immunol. Methods 220, 179–188.

9.	Nakano, H. and Yamane, T. (1998) Cell-free protein synthesis systems. Biotechnol. Adv. 16, 367–384.

10.	Jermutus, L., Ryabova, L.A., and Plückthun, A. (1998) Recent advances in producing and selecting functional proteins by using cell-free translation. Curr. Opin. Biotechnol. 9, 534–548.

11.	Mattheakis L. C., Bhatt R.R., and Dower W. J. (1994) An in vitro polysome display system for identifying ligands from very large peptide libraries. Proc. Natl. Acad. Sci. USA 91, 9022–9026.

12.	Hanes, J. and Plückthun, A. (1997) In vitro selection and evolution of functional proteins by using ribosome display. Proc. Natl. Acad. Sci. USA 94, 4937–4942.

13.	Hanes, J., Schaffitzel, C., Knappik, A., and Plückthun A. (2000) Picomolar affinity antibodies from a fully synthetic naïve library selected and evolved by ribosome display. Nature Biotechnol. 18, 1287–1292.

14.	Nemoto, N., Miyamoto-Sato, E., Hushimi, Y., and Yanagawa, H. (1997) In vitro virus: Bonding of mRNA bearing puromycin at the 3′-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro. FEBS Lett. 414, 405–408.

15.	Roberts, R.W. and Szostak, J. W. (1997) RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc. Natl. Acad. Sci. USA 94, 12,297–12,302.

16.	Tawfik, D.S. and Griffiths, A. D. (1998) Man-made cell-like compartments for molecular evolution. Nature Biotechnol. 16, 652–656.

17.	Griffiths, A. D. and Tawfik, D. S. (2003) Directed evolution of an extremely fast phosphotriesterase by in vitro compartmentalization. EMBO 22, 24–35.

18.	Martemyanov, K. A., Spirin, A.S., and Gudkov, A. T. (1997) Direct expression of PCR products in a cell-free transcription/translation system: synthesis of antibacterial peptide cecropin. FEBS Lett. 414, 268–270.

19.	Ohuchi, S., Nakano, H., and Yamane, T. (1998) In vitro method for the generation of protein libraries using PCR amplification of a single DNA molecule and coupled transcription/translation. Nucleic Acids Res. 26, 4339–4346.

20.	Nakano, H., Kobayashi, K., Ohuchi, S., Sekiguchi, S., and Yamane, T. (2000) Single-step single-molecule PCR of DNA with a homo-priming sequence using a single primer and hot-startable DNA polymerase. J. Biosci. Bioeng. 90, 456–458.

21.	Rungpragayphan, S., Kawarasaki, Y., Imaeda, T., Kohda, K., Nakano, H., and Yamane, T. (2002) High-throughput, cloning-independent protein library construction by combining single-molecule DNA amplification with in vitro expression. J. Mol. Biol. 318, 395–405.

22.	Rungpragayphan, S., Nakano, H., and Yamane, T. (2003) PCR-linked in vitro expression: a novel system for high-throughput construction and screening of protein libraries. FEBS Lett. 540, 147–150.

23.	Nakano, H., Kawarasaki, Y., and Yamane, T. (2004) Cell-free protein synthesis systems: Increasing their performance and applications. Adv. Biochem. Engin/Biotechnol. 90, 135–149.

24.	Koga, Y., Kobayashi, K., Yang, J., Nakano, H., and Yamane, T. (2002) In vitro construction and screening of a Burkholderia cepacia lipase library using single-molecule PCR and cell-free protein synthesis. J. Biosci. Bioeng. 94, 84–86.

25.	Koga, Y., Kato, K., Nakano, H., and Yamane, T. (2003) Inverting enantioselectivity of Burkholderia cepacia KWI-56 lipase by combinatorial mutation and high-throughput screening using single-molecule PCR and in vitro expression. J. Mol. Biol. 331, 585–592.

26.	Miyazaki-Imamura, C., Oohira, K., Kitagawa, R., Nakano, H., Yamane, T., and Takahashi, H. (2003) Improvement of H₂O₂ stability of manganese peroxidase by combinatorial mutagenesis and high-throughput screening using in vitro expression with protein disulfide isomerase. Protein Eng. 16, 423–428.

27.	Rungpragayphan, S., Haba, M., Nakano, H., and Yamane, T. (2004) Rapid screening for affinity-improved scFvs by means of single-molecule-PCR-linked in vitro expression. J. Mol. Catalysis B: Enzymatic 28, 223–228.

28.	Brownie, J., Shawcross, S., Theaker, J., et al. (1997) The elimination of primer-dimer accumulation in PCR. Nucleic Acids Res. 25, 3235–3241.

29.	Stephens, J. C., Rogers, J., and Ruano, G. (1990) Theoretical underpinning of the single-molecule-dilution (SMD) method of direct haplotype resolution. Am. J. Hum. Genet. 46, 1149–1155.

30.	Sambrook, J. and Russell, D. W. (2001) Molecular Cloning: A Laboratory annual, 3rd ed. Cold Spring Harbor Laboratory Press, NY.

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