Abstract
A wide variety of random- and site-directed mutagenesis techniques have been developed to investigate the structure–function relationship in proteins and intergenic regions like promoter sequences. Similar techniques can be employed to optimize protein properties like enantioselectivity, substrate specificity, and stability in a directed evolution approach. Due to the tremendous genetic diversity that is created by common random-mutagenesis methods, directed evolution techniques usually require the time-consuming and cumbersome screening of large numbers of variants. A gene-scanning saturation-mutagenesis approach represents one efficient way to limit the screening effort by reducing the created genetic diversity. In structure/function studies often a similar method, e.g., alanine- or arginine-scanning mutagenesis, is used to probe the role of specific amino acids in a protein. Here, we present a standardized mutagenesis strategy that can speed up the process of scanning whole proteins for structure/function studies and, furthermore, allows for the fast and efficient generation of gene-scanning saturation-mutagenesis libraries to be used in the directed evolution of enzyme functions and properties. The described method uses automated computer-assisted oligonucleotide design, and a two-step PCR-mutagenesis protocol to amplify site-specifically mutated circular plasmids that can be directly transformed in Escherichia coli expression strains.
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References
Valeva A, Siegel I, Wylenzek M et al (2008) Putative identification of an amphipathic alpha-helical sequence in hemolysin of Escherichia coli (HlyA) involved in transmembrane pore formation. Biol Chem 389:1201–1207
Hosaka Y, Iwata M, Kamiya N et al (2007) Mutational analysis of block and facilitation of HERG current by a class III anti-arrhythmic agent, nifekalant. Channels (Austin) 1:198–208
Shao Y, Feldman-Cohen LS, Osuna R (2008) Functional characterization of the Escherichia coli Fis-DNA binding sequence. J Mol Biol 376:771–785
Toloue MM, Woolwine Y, Karcz JA, Kasperek EM, Nicholson BJ, Skerrett IM (2008) Site-directed mutagenesis reveals putative regions of protein interaction within the transmembrane domains of connexins. Cell Commun Adhes 15:95–105
Sen J, Jacobs A, Caffrey M (2008) Role of the HIV gp120 conserved domain 5 in processing and viral entry. Biochemistry 47:7788–7795
Weis R, Gaisberger R, Gruber K, Glieder A (2007) Serine scanning: a tool to prove the consequences of N-glycosylation of proteins. J Biotechnol 129:50–61
Zupnick A, Prives C (2006) Mutational analysis of the p53 core domain L1 loop. J Biol Chem 281:20464–20473
Wong TS, Zhurina D, Schwaneberg U (2006) The diversity challenge in directed protein evolution. Comb Chem High Throughput Screen 9:271–288
Brissos V, Eggert T, Cabral JM, Jaeger KE (2008) Improving activity and stability of cutinase towards the anionic detergent AOT by complete saturation mutagenesis. Protein Eng Des Sel 21:387–393
Funke SA, Otte N, Eggert T, Bocola M, Jaeger KE, Thiel W (2005) Combination of computational prescreening and experimental library construction can accelerate enzyme optimization by directed evolution. Protein Eng Des Sel 18:509–514
Eggert T, Funke SA, Andexer JN, Reetz MT, Jaeger KE (2009) Evolution of Enantioselective Bacillus subtilis Lipase. In: Lutz S, Bornscheuer UT (eds) Protein engineering handbook. Wiley-VCH, Weinheim, pp 441–451
Barettino D, Feigenbutz M, Valcarcel R, Stunnenberg HG (1994) Improved method for PCR-mediated site-directed mutagenesis. Nucleic Acids Res 22:541–542
Miyazaki K, Takenouchi M (2002) Creating random mutagenesis libraries using megaprimer PCR of whole plasmid. Biotechniques 33:1033–1038
Krauss U, Eggert T (2005) insilico.mutagenesis: a primer selection tool designed for sequence scanning applications used in directed evolution experiments. Biotechniques 39:679–682
Lowe T, Sharefkin J, Yang SQ, Dieffenbach CW (1990) A computer program for selection of oligonucleotide primers for polymerase chain reactions. Nucleic Acids Res 18:1757–1761
Ling MM, Robinson BH (1997) Approaches to DNA mutagenesis: an overview. Anal Biochem 254:157–178
Sambrook J, Russell D (2001) Molecular cloning a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Breslauer KJ, Frank R, Blocker H, Marky LA (1986) Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci U S A 83:3746–3750
Allawi HT, SantaLucia J Jr (1997) Thermodyna-mics and NMR of internal G.T mismatches in DNA. Biochemistry 36:10581–10594
Bichet A, Bureik M, Lenz N, Bernhardt R (2004) The “Bringer” strategy: a very fast and highly efficient method for construction of mutant libraries by error-prone polymerase chain reaction of ring-closed plasmids. Appl Biochem Biotechnol 117:115–122
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Krauss, U., Jaeger, KE., Eggert, T. (2010). Rapid Sequence Scanning Mutagenesis Using In Silico Oligo Design and the Megaprimer PCR of Whole Plasmid Method (MegaWHOP). In: Braman, J. (eds) In Vitro Mutagenesis Protocols. Methods in Molecular Biology, vol 634. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-652-8_9
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DOI: https://doi.org/10.1007/978-1-60761-652-8_9
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