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.

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) (13,14) and the BL21(DE3)/Acya strain was prepared by P1 transduction as described (6). The pVEX-11 vector was a generous gift of V. Chaudhary (National Institutes of Health, Bethesda, MD) (15). BL21(DE3) cells were grown in LB broth, BL21(DE3)/pLysS and BL21(DE3)/pLysE were grown in LB broth with chloramphenicol (15 ,ug/ml), and all strains transformed with the pVEX constructs were grown in LB broth supplemented with ampicillin (50 ,ug/ml) (16).Preparation of the Polycistronic Expression Vector. A 1.1-kb Xba 1-HindIII fragment excised from pT7Ca (a gift from R. Maurer, University of Iowa) (7) was blunt-ended by incubation with the large fragment of DNA polymerase I (Klenow fragment) (17). This Ca fragment, containing a ribosome binding site (Shine-Dalgarno sequence) followed by the full-length Chinese hamster Ca cDNA, was blunt-end cloned (17) into the blunt-ended HindIII site of pVEXRI (6). An Asp718 site was generated in the vector by replacing the HindIII-Nde I fragment with the oligodeoxynucleotide seAbbreviations: PKA, cAMP-dependent protein kinase; IPTG, isopropyl -D-thiogalactopyranoside.

The regulatory subunit of a cGMP‐regulated protein kinase A of Trypanosoma brucei

“…Expression of the combined A and B domains resulted in much less protein (all insoluble). Expression of domain A alone proved impossible, despite much effort, in agreement with earlier observations that this domain is highly toxic for E. coli [37]. Thus ( Fig.…”

“…Expression of the combined A and B domains resulted in much less protein (all insoluble). Expression of domain A alone proved impossible, despite much effort, in agreement with earlier observations that this domain is highly toxic for E. coli [37]. Thus, domains A and B were expressed individually in the Drosophila cell line S2, under the control of a Cu 2+ ‐inducible metallothionein promoter.…”

Overexpression of Mouse Estrogen Receptor-β Decreases but Its Transactivation and Ligand Binding Domains Increase the Growth Characteristics of E. coli

“…Bacterial cells need energy for growth and maintenance of metabolic activity and their metabolic activity gets disrupted when a recombinant protein is overexpressed. Further, the overexpressions of FTNF gene and cAMP-dependent protein kinase also decrease bacterial growth [14]. Many reports suggest that disruption in metabolic load, underproduction of other components, maintenance and overexpression of recombinant proteins adversely affect the bacterial growth [15][16][17][18].…”

Phosphorylation and activation of a transducible recombinant form of human HSP20 in Escherichia coli

“…Several protein kinases and their subunits have been expressed in E. coli in both active and inactive forms, including cGMP-and cAMP dependent protein kinases, myosin light chain kinase, and casein kinase [20][21][22][23][24][25][26][27][28][29]. Full length bovine cGMP-dependent protein kinase has been expressed previously in E. coli using both T7 RNA polymerase and tac+ promoters [21].…”