Lookup NU author(s): Dr Tahar Taybi
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Plant phosphoenolpyruvate-carboxylase kinase (PEPC-kinase [PpcK]) is the smallest Ser/Thr kinase identified to date, having a molecular mass of ∼32,000. This novel, monomeric kinase is dedicated to the phosphorylation of plant PEPC, thereby regulating this target enzyme's activity and allosteric properties. Although several recombinant, non-fusion PpcK proteins have been produced recently in Escherichia coli, these are plagued by their high degree of insolubility. Here, we report the use of the native, E. coli NusA protein and a related E. coli expression vector (pET-43a(+) [Novagen]) for enhancing the solubility of this recalcitrant Ser/Thr kinase at least 10-fold by its production as a dual 6xHis-tagged NusA/McPpcK1 fusion protein, which accounts for ∼10% of the soluble protein fraction from induced cells. Capture of this fusion protein from the centrifuged cell extract by immobilized metal (Ni2+) affinity-chromatography, its "on-bead" cleavage by thrombin, and subsequent elution yielded milligram quantities of a "free," ∼36-kDa form of PpcK for further purification by fast-protein liquid chromatography on blue dextran-agarose or preparative SDS-PAGE. Steady-state kinetic analysis of the former, active preparation revealed that this dedicated kinase discriminates against neither various isoforms of plant PEPC nor certain mutant forms of recombinant C4 PEPC. Alternatively, the latter, electrophoretically homogeneous sample of the ∼36-kDa polypeptide was used as antigen for polyclonal-antibody production in rabbits. The antibodies against the recombinant McPpcK1 from Mesembryanthemum crystallinum cross-reacted on Western blots with an enriched preparation of the maize-leaf kinase, but not with the parent crude extract, thus directly documenting this protein's extremely low abundance in vivo. However, these antibodies were effective in immunoprecipitating 32P-based PpcK activity from crude, desalted extracts of maize leaves and soybean root-nodules. © 2003 Elsevier Science (USA). All rights reserved.
Author(s): Ermolova NV, Cushman MA, Taybi T, Condon SA, Cushman JC, Chollet R
Publication type: Article
Publication status: Published
Journal: Protein Expression and Purification
ISSN (print): 1046-5928
ISSN (electronic): 1096-0279
Publisher: Academic Press
PubMed id: 12729733
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