Recombinant production of the human complement factor 5a in Escherichia coli

Autor: Nausch,H; Huckauf,J; Koslowski,R; Meyer,U; Broer,I; Mischofsky,H
In:

African Journal of Biotechnology

Bandangabe: 11
Auflage: 81
Gesamttitelundnr1: Recombinant production of the human complement factor 5a in Escherichia coli
ISBN: DOI: 10.5897/AJB12.2258
Seite: 14685 - 14695
Jahr: 2012

Einordung:
Institut: Professur Agrobiotechnologie

Abstract:
Up to now, the human complement factor 5a (C5a) has only been produced in small quantities in Escherichia coli in a soluble, bioactive conformation, which is not suitable for commercial production systems. This stems from the extremely high instability of C5a, as well as its aggregation-prone nature. Therefore, we analyzed several different methods for optimizing the solubility and biological activity of C5a produced by E. coli. The solubility of C5a was efficiently improved by expressing it as a glutathione-S-transferase (GST) fusion protein and, to a lesser extent, by lowering the cultivation temperature. Neither reducing the inductor concentration (isopropylthio-β-galactoside, IPTG) of the T7lac promotor nor the concomitant overexpression of endogenous chaperones was effective. However, the biological activity of the protein was improved by the overexpression of chaperones together with cultivation at 22°C, while fusion to GST slightly reduced its activity. Consequently, low cultivation temperature and the overexpression of chaperones seem to be the optimal strategy for expression of appropriate amounts of soluble and functional C5a. These findings should be the basis for the transfer to large-scale fermentation. Using C5a as an example, we showed that strain engineering in combination with specific cultivation conditions improve the production of difficult-to-express proteins in appropriate amounts and in a functional conformation facilitating the commercial manufacturing under good manufacturing practices (GMP) conditions.

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