A fed-batch based cultivation mode in E.coli results in improved specific activity of a novel chimeric-truncated form of tissue plasminogen activator.

Fereidoun Mahboud (1) , Farzaneh Barkhordari (1) , Reza Moazami Godarzi (1) , Somayeh Enayati (1) , Fatemeh Davami (1)

1 Biotechnology Research Center

AIMS: A novel chimeric-truncated form of t-PA with improved fibrin affinity and resistance to PAI was successfully produced in CHO expression system during our previous studies. Considering advantages of prokaryotic expression systems, the aim in this study was to produce the novel protein in E.coli (BL21) strain and compare the protein potency in batch and fed-batch processes. METHODS AND RESULTS: The expression cassette for the novel t-PA was prepared in pET-28a(+). The E.coli expression procedure was compared in traditional batch and newly developed fed batch; EnBase® Flo system. The protein was purified in soluble format and potency results were identified using Chromolize t-PA assay kit. The fed-batch fermentation mode, coupled with a Ni-NTA affinity purification procedure under native condition resulted in higher amounts of soluble protein, and about a 30% of improvement in the specific activity of the resulted recombinant protein (46.66 IU mg(-1) ) compared to traditional batch mode (35.8 IU mg(-1) ). CONCLUSIONS: Considering the undeniable advantages of expression in the prokaryotic expression systems such as E.coli for recombinant protein production, applying alternative methods of cultivation is a promising approach. In this study, fed-batch cultivation methods showed the potential to replace miss folded formats of protein with proper folded, soluble form with improved potency. SIGNIFICANCE AND IMPACT OF STUDY: E.coli expression of recombinant proteins, still counts for nearly 40% of marketed biopharmaceuticals. The major drawback of this system is the lack of appropriate post translational modifications, which may cause potency loss/decline. Therefore, applying alternative methods of cultivation as investigated here is a promising approach to overcome potency decrease problem in this protein production system. © 2012The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

Keywords: Escherichia coli expression; fed-batch cultivation; specific activity; tissue-type plasminogen activator.; tissue-type plasminogen activator

Domains: Life Sciences [q-bio]; Biotechnology; Computer Science [cs]; Biotechnology

Fereidoun Mahboud, Farzaneh Barkhordari, Reza Moazami Godarzi, Somayeh Enayati, Fatemeh Davami. A fed-batch based cultivation mode in E.coli results in improved specific activity of a novel chimeric-truncated form of tissue plasminogen activator.. Journal of Applied Microbiology, 2012, epub ahead of print. ⟨10.1111/jam.12059⟩. ⟨pasteur-00753198⟩