Overexpression and purification of folded domain of prostate cancer related proteins MSMB and PSA

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• 作者：Mohini Tiwary ; Nipanshu Agarwal ; Amit Dinda ; Subhash C. Yadav
• 刊名：Molecular Biology Reports
• 出版年：2016
• 出版时间：May 2016
• 年：2016
• 卷：43
• 期：5
• 页码：349-358
• 全文大小：1,657 KB
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Life Sciences
  Animal Anatomy, Morphology and Histology
  Animal Biochemistry
  
• 出版者：Springer Netherlands
• ISSN：1573-4978
• 卷排序：43

文摘

Overexpression of domains of a human protein using recombinant DNA technology has been challenging because individual domains intend to accumulate as non-soluble aggregate when expressed separately. Studies on identifying right sequences for a domain to be able to fold independently may help understand the folding pattern and underlying protein-engineering events to isolate the functional domains of a protein. In this report, individual domains of prostate cancer related biomarkers; MSMB and PSA were overexpressed in bacterial system and purified in their folded forms using affinity chromatography. The western blotting experiment using domain specific antibodies further confirmed these proteins. The designed nucleotide sequences domains were truncated using fold index software and folding were predicted by phyre2 and I-TASSER software. Other parameters were optimized for their overexpression and purification using Co-NTA affinity chromatography. Purified domains of each protein showed secondary structures such as α + β type for PSA, α/β and β type for the each domains of PSA and MSMB respectively. This is the first report on producing PSA and MSMB individual domains in functional folded forms. This study may help produce the folded domain of many such proteins to be used for better diagnostic purpose.KeywordsPSAMSMBDomainsFoldingWestern blottingRecombinant protein