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[Cloning, sequencing, expression, and primary identification of recombinant mouse protein kinase CK2 alpha subunit]
Xiao-wen Chen1, Xin-guang Liu, Ke-qin Zheng
1Institute of Biochemistry and Molecular Biology, Guangdong Medical College, Zhanjiang, 524023, P. R. China.
Background & 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.
Insights
Researchers successfully cloned and expressed the mouse protein kinase CK2 alpha subunit, a key enzyme in cancer. This recombinant protein is crucial for further studies on CK2
Area of Science:
- Molecular Biology
- Biochemistry
- Oncology
Background:
- Protein kinase CK2 (CK2) is a conserved eukaryotic enzyme.
- Elevated CK2 levels are linked to oncogenesis in various tumors.
- Understanding CK2 structure and function is vital for cancer research.
Purpose of the Study:
- To construct and express a recombinant plasmid encoding the mouse CK2 alpha subunit.
- To facilitate further research into the structure and function of CK2.
- To develop a tool for studying CK2's role in cancer.
Main Methods:
- Mouse CK2 alpha subunit cDNA was amplified using RT-PCR.
- The cDNA was cloned into the pT7-7 expression vector.
- Recombinant plasmid was transformed into E. coli and expressed using IPTG induction.
- Protein expression was confirmed via Western blotting.
Main Results:
- A 100% positive transformation rate was achieved.
- Sequencing confirmed the integrity of the cloned mouse CK2 alpha subunit cDNA.
- A 42 kDa protein was overexpressed and confirmed by Western blot.
- The recombinant protein showed specific reactivity with anti-human CK2 alpha antibody.
Conclusions:
- Successfully cloned and expressed the recombinant mouse protein kinase CK2 alpha subunit.
- The generated recombinant plasmid and protein are suitable for further investigation.
- This work provides a foundation for studying CK2's oncogenic roles.