NDLI: Expression of Chinese hamster cAMP-dependent protein kinase in Escherichia coli results in growth inhibition of bacterial cells: a model system for the rapid screening of mutant type I regulatory subunits.

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Expression of Chinese hamster cAMP-dependent protein kinase in Escherichia coli results in growth inhibition of bacterial cells: a model system for the rapid screening of mutant type I regulatory subunits.

Content Provider	PubMed Central
Author	Gosse, M. E. Padmanabhan, A. Fleischmann, R. D. Gottesman, M. M.
Abstract	The regulatory and catalytic subunits of cAMP-dependent protein kinase (PKA) were coexpressed within the same bacterial cell using a polycistronic bacterial T7 expression vector encoding Chinese hamster cDNAs for the type I regulatory (RI) and catalytic alpha (C alpha) subunits of PKA. Basal expression of active RI/C alpha holoenzyme in the BL21(DE3) strain of Escherichia coli caused severe growth inhibition resulting in extremely small colony size. Several lines of evidence demonstrate that this growth inhibition requires active PKA subunits and cAMP: (i) this phenotype is dependent on cAMP since it is not seen in a strain lacking adenylyl cyclase activity, but the growth rate of these transformants is slower when exogenous cAMP is added; (ii) normal growth occurs when wild-type RI cDNA is replaced by a mutant RI cDNA encoding a RI protein with reduced cAMP binding; and (iii) the growth-inhibited phenotype of the transformed BL21(DE3) cells requires soluble, active C alpha protein. Holoenzyme expressed in bacteria is activated by cAMP, which stimulates phosphorylation of an endogenous 50-kDa protein that is missing in four host mutants selected for normal growth after transformation with PKA holoenzyme. A mutant RI cDNA library was generated by PCR random mutagenesis and screened by polycistronic expression in BL21(DE3) cells. The RI cDNA sequence from one revertant has base-pair substitutions creating two amino acid substitutions within the cAMP binding sites. The coexpression of the RI/C alpha subunits in BL21(DE3) bacterial cells provides a system for rapidly selecting mutations in the RI subunits of PKA.
Starting Page	8159
File Format	PDF
ISSN	10916490
e-ISSN	10916490
Journal	Proceedings of the National Academy of Sciences of the United States of America
Issue Number	17
Volume Number	90
Language	English
Publisher Date	1993-09-01
Access Restriction	Open
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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