Biomaterial Database

[Cloning, sequencing, expression, and primary identification of recombinant mouse protein kinase CK2 alpha subunit]. 2002

Xiao-wen Chen, and Xin-guang Liu, and Ke-qin Zheng, and Nian-ci Liang

Institute of Biochemistry and Molecular Biology, Guangdong Medical College, Zhanjiang, 524023, P. R. China.

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.

UI	MeSH Term	Description	Entries
D011994	Recombinant Proteins	Proteins prepared by recombinant DNA technology.	Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D003001	Cloning, Molecular	The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells.	Molecular Cloning
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D000818	Animals	Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA.	Animal,Metazoa,Animalia
D015153	Blotting, Western	Identification of proteins or peptides that have been electrophoretically separated by blot transferring from the electrophoresis gel to strips of nitrocellulose paper, followed by labeling with antibody probes.	Immunoblotting, Western,Western Blotting,Western Immunoblotting,Blot, Western,Immunoblot, Western,Western Blot,Western Immunoblot,Blots, Western,Blottings, Western,Immunoblots, Western,Immunoblottings, Western,Western Blots,Western Blottings,Western Immunoblots,Western Immunoblottings
D015870	Gene Expression	The phenotypic manifestation of a gene or genes by the processes of GENETIC TRANSCRIPTION and GENETIC TRANSLATION.	Expression, Gene,Expressions, Gene,Gene Expressions
D016133	Polymerase Chain Reaction	In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships.	Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain
D016475	3T3 Cells	Cell lines whose original growing procedure consisted being transferred (T) every 3 days and plated at 300,000 cells per plate (J Cell Biol 17:299-313, 1963). Lines have been developed using several different strains of mice. Tissues are usually fibroblasts derived from mouse embryos but other types and sources have been developed as well. The 3T3 lines are valuable in vitro host systems for oncogenic virus transformation studies, since 3T3 cells possess a high sensitivity to CONTACT INHIBITION.	3T3 Cell,Cell, 3T3,Cells, 3T3
D017346	Protein Serine-Threonine Kinases	A group of enzymes that catalyzes the phosphorylation of serine or threonine residues in proteins, with ATP or other nucleotides as phosphate donors.	Protein-Serine-Threonine Kinases,Serine-Threonine Protein Kinase,Serine-Threonine Protein Kinases,Protein-Serine Kinase,Protein-Serine-Threonine Kinase,Protein-Threonine Kinase,Serine Kinase,Serine-Threonine Kinase,Serine-Threonine Kinases,Threonine Kinase,Kinase, Protein-Serine,Kinase, Protein-Serine-Threonine,Kinase, Protein-Threonine,Kinase, Serine-Threonine,Kinases, Protein Serine-Threonine,Kinases, Protein-Serine-Threonine,Kinases, Serine-Threonine,Protein Kinase, Serine-Threonine,Protein Kinases, Serine-Threonine,Protein Serine Kinase,Protein Serine Threonine Kinase,Protein Serine Threonine Kinases,Protein Threonine Kinase,Serine Threonine Kinase,Serine Threonine Kinases,Serine Threonine Protein Kinase,Serine Threonine Protein Kinases
D047390	Casein Kinase II	A ubiquitous casein kinase that is comprised of two distinct catalytic subunits and dimeric regulatory subunit. Casein kinase II has been shown to phosphorylate a large number of substrates, many of which are proteins involved in the regulation of gene expression.	Casein Kinase 2,Casein Kinase 2 alpha Subunit,Casein Kinase 2 alpha' Subunit,Casein Kinase 2 beta Subunit,Casein Kinase 2alpha,Casein Kinase 2alpha',Casein Kinase TS,Casein Kinase-2,Protein Kinase CK2,Protein Kinase CKII,2alpha', Casein Kinase,Kinase 2alpha', Casein,Kinase CKII, Protein