caa a22 4500 60549827X CHVBK 20210128100545.0 cr unu---uuuuu 210128e20150801xx s 000 0 eng 10.1007/s00253-015-6751-6 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6751-6 Expression and rapid purification of recombinant biologically active ovine growth hormone with DsbA targeting to Escherichia coli inner membrane [Elektronische Daten] [Faiza Durrani, Roquyya Gul, Saima Sadaf, Muhammad Akhtar] This study shows expression of recombinant ovine growth hormone (roGH) and targeting to the inner membrane using signal sequence, DsbA, in Escherichia coli (E. coli) cell. Factors such as temperature, IPTG induction, and expression conditions were studied and show diverse optical density with different media compositions. The optimum expression level of roGH in terrific broth medium was at 25°C on induction with 20μM IPTG in early logarithmic phase. SDS-PAGE analysis of expression and subcellular fractions of recombinant constructs revealed the translocation of roGH to the inner membrane of E. coli with DsbA signal sequence at the N terminus of roGH. The protein was easily solubilized by 40% acetonitrile with ~90% purity and was identified by Western blot, and analysis on MALDI-TOF/TOF confirmed a size of 21,059Da. Relatively high soluble protein yield of 65.3mg/L of roGH was obtained. The biological function of roGH was confirmed by HeLa cell line proliferation. This is the first study describing achievement of biologically active soluble roGH targeted to the inner membrane of E. coli and rapid purification with high yield. Springer-Verlag Berlin Heidelberg, 2015 DsbA signal sequence nationallicence HeLa cell lines nationallicence Ovine growth hormone nationallicence MALDI-TOF/TOF nationallicence Durrani Faiza School of Biological Sciences, University of the Punjab, Lahore, Pakistan aut Gul Roquyya Centre for Research in Molecular Medicine, The University of Lahore, 1-km Defense Road, Bhobatian Chowk, Lahore, Pakistan aut Sadaf Saima Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore, Pakistan aut Akhtar Muhammad School of Biological Sciences, University of the Punjab, Lahore, Pakistan aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/16(2015-08-01), 6791-6801 0175-7598 99:16<6791 2015 99 253 https://doi.org/10.1007/s00253-015-6751-6 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6751-6 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Durrani Faiza School of Biological Sciences, University of the Punjab, Lahore, Pakistan aut NATIONALLICENCE 700 1- Gul Roquyya Centre for Research in Molecular Medicine, The University of Lahore, 1-km Defense Road, Bhobatian Chowk, Lahore, Pakistan aut NATIONALLICENCE 700 1- Sadaf Saima Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore, Pakistan aut NATIONALLICENCE 700 1- Akhtar Muhammad School of Biological Sciences, University of the Punjab, Lahore, Pakistan aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/16(2015-08-01), 6791-6801 0175-7598 99:16<6791 2015 99 253