Expression and characterization of phosphorylated recombinant human beta-casein in Escherichia coli

Specific serine and threonine residues of recombinant human beta-casein produced in Escherichia coli were shown to be phosphorylated in vivo when human casein kinase II was coexpressed in the same plasmid. All of the phosphorylated forms found in the native protein were also detected in the recombin...

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Bibliographic Details
Published in:Protein expression and purification 1997-07, Vol.10 (2), p.202
Main Authors: Thurmond, J M, Hards, R G, Seipelt, C T, Leonard, A E, Hansson, L, Strömqvist, M, Byström, M, Enquist, K, Xu, B C, Kopchick, J J, Mukerji, P
Format: Article
Language:English
Subjects:
Casein Kinase II
Caseins - biosynthesis
Caseins - chemistry
Caseins - genetics
Caseins - isolation & purification
Coenzymes - analysis
Coenzymes - genetics
Humans
Mass Spectrometry
Mutagenesis, Insertional
Phosphorylation
Plasmids - genetics
Protein-Serine-Threonine Kinases - analysis
Protein-Serine-Threonine Kinases - genetics
Recombinant Proteins - biosynthesis
Recombinant Proteins - chemistry
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Summary:Specific serine and threonine residues of recombinant human beta-casein produced in Escherichia coli were shown to be phosphorylated in vivo when human casein kinase II was coexpressed in the same plasmid. All of the phosphorylated forms found in the native protein were also detected in the recombinant protein. The phosphorylation of recombinant human beta-casein was confirmed by immunoblots, fast protein liquid chromatography, urea-polyacrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis, and liquid chromatography-mass spectrometry. The results indicate that the substrate specificity of casein kinase II in vivo was unaffected in its recombinant form. This is the first demonstration of in vivo phosphorylation of specific residues of a multiphosphorylated protein produced in E. coli with a single plasmid.
ISSN:1046-5928
DOI:10.1006/prep.1997.0737