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.
Molecular biology reports. 2016 Apr 01 [Epub ahead of print]
Mohini Tiwary, Nipanshu Agarwal, Amit Dinda, Subhash C Yadav
Department of Pathology, All India Institute of Medical Sciences (AIIMS), New Delhi, 110029, India., TERI-Deakin Nano Biotechnology Centre, The Energy and Resources Institute, Darbari Seth Block, IHC Complex, Lodhi Road, New Delhi, 110003, India., Department of Pathology, All India Institute of Medical Sciences (AIIMS), New Delhi, 110029, India. TERI-Deakin Nano Biotechnology Centre, The Energy and Resources Institute, Darbari Seth Block, IHC Complex, Lodhi Road, New Delhi, 110003, India.