title: A fed-batch based cultivation mode in Escherichia coli results in improved specific activity of a novel chimeric-truncated form of tissue plasminogen activator.
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: F
authors.initials: F
authors.lastname: Mahboudi
authors.forename: F
authors.initials: F
authors.lastname: Barkhordari
authors.forename: R M
authors.initials: RM
authors.lastname: Godarzi
authors.forename: S
authors.initials: S
authors.lastname: Enayati
authors.forename: F
authors.initials: F
authors.lastname: Davami
mesh_terms: D000818:Animals; D061249:Batch Cell Culture Techniques; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D010959:Tissue Plasminogen Activator / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology
chemical_list: D005343:Fibrinolytic Agents; D011993:Recombinant Fusion Proteins; D010959:Tissue Plasminogen Activator