title: In vitro recombination of non-homologous genes can result in gene fusions that confer a switching phenotype to cells.
abstract: Regulation of protein activity is central to the complexity of life. The ability to regulate protein activity through exogenously added molecules has biotechnological/biomedical applications and offers tools for basic science. Such regulation can be achieved by establishing a means to modulate the specific activity of the protein (i.e. allostery). An alternative strategy for intracellular regulation of protein activity is to control the amount of protein through effects on its production, accumulation, and degradation. We have previously demonstrated that the non-homologous recombination of the genes encoding maltose binding protein (MBP) and TEM1 β-lactamase (BLA) can result in fusion proteins in which β-lactamase enzyme activity is allosterically regulated by maltose. Here, through use of a two-tiered genetic selection scheme, we demonstrate that such recombination can result in genes that confer maltose-dependent resistance to β-lactam even though they do not encode allosteric enzymes. These 'phenotypic switch' genes encode fusion proteins whose accumulation is a result of a specific interaction with maltose. Phenotypic switches represent an important class of proteins for basic science and biotechnological applications in vivo.
authors.affiliation: Department of Chemical & Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, United States of America.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Heins
authors.forename: Jay H
authors.initials: JH
authors.lastname: Choi
authors.forename: Takayuki
authors.initials: T
authors.lastname: Sohka
authors.forename: Marc
authors.initials: M
authors.lastname: Ostermeier
mesh_terms: D000494:Allosteric Regulation; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D050939:Gene Fusion / Q000235:genetics / Q000502:physiology; D008320:Maltose / Q000494:pharmacology; D058619:Maltose-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011995:Recombination, Genetic / Q000235:genetics*; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
chemical_list: D058619:Maltose-Binding Proteins; D008320:Maltose; D001618:beta-Lactamases
references.citation: Curr Opin Struct Biol. 2009 Aug;19(4):442-8
references.pmid: 19473830
references.citation: Methods Enzymol. 1986;131:266-80
references.pmid: 3773761
references.citation: Biochem J. 1995 Jun 15;308 ( Pt 3):859-64
references.pmid: 8948443
references.citation: Protein Sci. 1997 Jan;6(1):141-6
references.pmid: 9007986
references.citation: J Mol Biol. 2004 Feb 6;336(1):263-73
references.pmid: 14741221
references.citation: Proc Natl Acad Sci U S A. 2009 Jun 23;106(25):10135-40
references.pmid: 19502423
references.citation: Biophys J. 2005 Nov;89(5):2988-97
references.pmid: 16113118
references.citation: Proc Natl Acad Sci U S A. 2011 Sep 27;108(39):16206-11
references.pmid: 21930952
references.citation: J Mol Biol. 1994 Dec 16;244(5):625-39
references.pmid: 7990143
references.citation: Proc Natl Acad Sci U S A. 2005 Aug 9;102(32):11224-9
references.pmid: 16061816
references.citation: J Biol Chem. 1984 Apr 25;259(8):5327-32
references.pmid: 6425288
references.citation: J Mol Biol. 1990 Jul 5;214(1):337-52
references.pmid: 2196376
references.citation: Proc Natl Acad Sci U S A. 2003 Oct 28;100(22):12700-5
references.pmid: 14530390
references.citation: Methods Enzymol. 2007;428:459-86
references.pmid: 17875434
references.citation: EMBO J. 1994 Mar 1;13(5):1226-34
references.pmid: 8131752
references.citation: Mol Cell. 2009 Dec 11;36(5):861-71
references.pmid: 20005848
references.citation: J Bacteriol. 1980 Aug;143(2):761-71
references.pmid: 6259122
references.citation: J Biol Chem. 1998 Feb 20;273(8):4323-8
references.pmid: 9468480
references.citation: Chem Biol. 2004 Nov;11(11):1483-7
references.pmid: 15555998
references.citation: J Mol Biol. 1991 Jul 20;220(2):507-30
references.pmid: 1856871
references.citation: Protein Eng Des Sel. 2007 Jul;20(7):315-8
references.pmid: 17575283
references.citation: Protein Expr Purif. 2005 May;41(1):207-34
references.pmid: 15915565