Effect of Twine-arginine Translocation-signaling Fusion System and Chaperones Co-expression on Secretory Expression of Somatropin

Abstract

Background:Twine-arginine translocation (TAT) system is one of the exporting systems in Escherichia coli which could transport fully/semi-correctly folded proteins outside the reductive cytoplasmic space. In combination with co-expression with a chaperone system, the correctly folded proteins could be transported to oxidative periplasmic space and culture media to pass the main limitations in E. coli expression system such as misfolding and inclusion body formation.Materials and Methods:To study the effectiveness of signaling sequences and chaperone co-expression on the translocation of expressed protein, somatropin was selected as the target. Two common signal sequences in TAT system (TorA and SufI) were added at the N-terminal of somatropin and the cassettes were co-expressed in E. coli BL21 (DE3) by a chaperone team including DnaK/J-GrpeE.Results:The expression pattern studies including Western blotting and sodium dodecyl sulfate polyacrylamide gel electrophoresis confirmed that somatropin is expressed in two cassettes. However, the pattern was different for two signaling sequences.Conclusion:The results confirmed that the approach of using TAT-signaling sequences and co-expression with the chaperone team could enhance translocation of protein to periplasmic space and culture media compared to control groups. Western blotting results showed that the signal sequence TorA could transport more expressed proteins to the periplasmic space and culture media in comparison with SufI. However, there was a considerable amount of human growth hormone in the cytoplasm which could not be transported outside the cytoplasmic space.

Growth hormone receptor agonists and antagonists: From protein expression and purification to long‐acting formulations

“…The signal peptide is cleaved by a signal peptidase during or shortly after substrate translocation, and the mature protein is released on the trans-side of the membrane (Freudl, 2018). Periplasmic secretion has been widely used for the production of rhGH (Alanen et al, 2015;Amaranto et al, 2021;Bagherinejad et al, 2016Bagherinejad et al, , 2018Becker & Hsiung, 1986;Browning et al, 2017;Chang et al, 1987;Chang et al, 1989;Ghorpade & Garg, 1993;Gray et al, 1985;Guerrero Montero et al, 2019;Jeiranikhameneh et al, 2017;Li et al, 2004;Matos et al, 2014;Menezes et al, 2017;Perez-Perez et al, 2020;Rigi et al, 2021;Soares et al, 2003;Soares et al, 2008;Sockolosky & Szoka, 2013;Teresa et al, 2000;Uchida et al, 1997;Zamani et al, 2016;Zhou et al, 2021). A common approach is to fuse the signal sequence to the N-terminus of rhGH for transport, and a His-tag to the C-terminus for subsequent purification.…”