Experimental Analysis of E2BB (LTIIb) Signal Peptide in Secretory Production of Reteplase in Escherichia coli

Abstract

Recombinant reteplase is the truncated form of tissue plasminogen activator. Signal peptides play a pivotal role in the secretion of recombinant proteins. This study aimed to evaluate the effect of LTIIb signal peptide on the recombinant reteplase secretion in Escherichia coli. In the current study, cloning and expression of reteplase coding sequence with and without sequence corresponds to the peptide signal using pET 28a system was performed and its recombinant protein expression in E. coli BL21 (DE3) was induced by adding isopropyl β-d-1-thiogalactopyranoside (IPTG) to the final concentrations 0.1 mM. The recombinant reteplase expression was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot assay. The activity of reteplase in periplasmic fraction and supernatant was measured by colorimetric assay. Our results confirm that high levels of reteplase protein are expressed as inclusion bodies. In the presence of peptide signal LTIIb, in periplasmic fraction, reteplase activity was negligible compared to the reteplase standard. No activity observed for reteplase in the periplasmic and extracellular fraction. The comparison between results obtained for LTIIb-reteplase with those to reteplase revealed that LTIIb signal peptide is not the proper candidate for the secretory production of reteplase.

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Acknowledgements

This work has been supported by the Research and Technology Deputy of Shiraz University of Medical Sciences, Shiraz, Iran.

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Correspondence to Pegah Mousavi.

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Mousavi, P., Morowvat, M.H., Mostafavi-Pour, Z. et al. Experimental Analysis of E2BB (LTIIb) Signal Peptide in Secretory Production of Reteplase in Escherichia coli. Int J Pept Res Ther 27, 209–218 (2021). https://doi.org/10.1007/s10989-020-10059-x

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Keywords

  • Signal peptide
  • Secretion
  • Cytoplasmic expression
  • Reteplase
  • Escherichia coli
  • LTIIb (E2BB)