[Cloning, sequencing, expression, and primary identification of recombinant mouse protein kinase CK2 alpha subunit].
OBJECTIVE
Protein kinase CK2 is a highly conserved and ubiquitous eukaryotic serine/threonine kinase that is elevated and can serve as an oncogene in many tumor cells. To further research the structure and function of CK2, this study was designed to construct, express, and preliminarily identify a recombinant expression plasmid which contains the cDNA encoding mouse protein kinase CK2 alpha subunit.


METHODS
The aimed cDNA was obtained from NIH 3T3 mouse fibroblasts by RT-PCR. Nde I/BamH I-digested PCR product was directly cloned into pT7-7 expression vector which had been digested by Nde I/BamH I and dephosphorylated by calf intestinal alkaline phosphatase in advance. After E. coli DH5 alpha was transformed with the recombinant DNA by CaCl2 method, transformants were obtained. The positive clones were screened out primarily by gel electrophoresis, and then analyzed by digesting with restriction enzyme. Four positive clones were selected at random and sequenced respectively. The correct recombinant plasmid was transformed into E. coli BL21(DE3) and then expressed by inducing with IPTG. The products were identified with Western blotting.


RESULTS
The positive rate of transformants was 100%. The results of restriction analysis indicated that DNA band size of the insert fragment and recombinant plasmid were consistent with theoretically predicated values. The sequencing results showed one of the four clones possessed the cDNA sequence which has no mutation in the processing of PCR, which was termed as pTMCKA. One protein with molecular mass of 42 kDa was overexpressed by inducing with IPTG. The Western blot results confirmed that the recombinant product could specially react with antibody against human CK2 alpha subunit.


CONCLUSIONS
The authors have successfully cloned and expressed recombinant mouse protein kinase CK2 alpha subunit in this experiment.
Chen
Xiao-wen
XW
Institute of Biochemistry and Molecular Biology, Guangdong Medical College, Zhanjiang, 524023, P. R. China.
Liu
Xin-guang
XG
Zheng
Ke-qin
KQ
Liang
Nian-ci
NC
D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D047390:Casein Kinase II; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D051379:Mice; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis
D018076:DNA, Complementary; D011994:Recombinant Proteins; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases