ORIGINAL ARTICLE A fed-batch based cultivation mode in Escherichia coli results in improved specific activity of a novel chimeric -truncated form of tissue plasminogen activator F. Mahboudi, F. Barkhordari, R.M. Godarzi, S. Enayati and F. Davami Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran Keywords Escherichia coli expression, fed-batch cultivation, specific activity, tissue-type plasminogen activator. Correspondence Fatemeh Davami, Biotechnology Research Center, Pasteur Institute of Iran (IPI), No. 358, 12th Farvardin Ave, Jomhhoori St, Tehran 1316943551, Iran. E-mail: f_davami@pasteur.ac.ir 2013/1633: received 12 September 2012, revised 29 October 2012 and accepted 29 October 2012 doi:10.1111/jam.12059 Abstract Aims: A novel chimeric-truncated form of tissue-type plasminogen activator (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 Escherichia 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 (46Á66 IU mg À1 ) compared to traditional batch mode (35Á8 IU mg À1 ). 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 the Study: Escherichia 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. Introduction Tissue-type plasminogen activator (t-PA) is a glycopro- tein consisting of 527 amino acid residues (72 kDa), which is FDA approved for the treatment of myocardial infarction. Enhanced activity in the presence of fibrin, that is, fibrin-specific plasminogen activation, is the major advantage of t-PA over other thrombolytic agents. Bowes melanoma cells were the first source of t-PA as a therapeutic (Griffiths and Electricwala 1987). To meet the need for sufficient production of the drug through a robust industrial process, recombinant t-PA (rt-PA) was produced in mammalian cells (Lubiniecki et al. 1990; Cartwright 1992). However, low volumetric yields of the recombinant protein, long cultivation times and the need for expensive bioreactors and medium components are 364 Journal of Applied Microbiology 114, 364--372 © 2012 The Society for Applied Microbiology Journal of Applied Microbiology ISSN 1364-5072