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 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.
Thurmond
J M
JM
Abbott Laboratories, Ross Products Division, Columbus, Ohio 43219, USA.
Hards
R G
RG
Seipelt
C T
CT
Leonard
A E
AE
Hansson
L
L
Strömqvist
M
M
Byström
M
M
Enquist
K
K
Xu
B C
BC
Kopchick
J J
JJ
Mukerji
P
P
D047390:Casein Kinase II; D002364:Caseins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003067:Coenzymes / Q000032:analysis / Q000235:genetics; D006801:Humans; D013058:Mass Spectrometry; D016254:Mutagenesis, Insertional; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry
D002364:Caseins; D003067:Coenzymes; D011994:Recombinant Proteins; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases