title: Batch production of a silk-elastin-like protein in E. coli BL21(DE3): key parameters for optimisation.
abstract: BACKGROUND
  Silk-elastin-like proteins (SELPs) combining the physicochemical and biological properties of silk and elastin have a high potential for use in the pharmaceutical, regenerative medicine and materials fields. Their development for use is however restrained by their production levels. Here we describe the batch production optimisation for a novel recently described SELP in the pET-E. coli BL21(DE3) expression system. Both a comprehensive empirical approach examining all process variables (media, induction time and period, temperature, pH, aeration and agitation) and a detailed characterisation of the bioprocess were carried out in an attempt to maximise production with this system.
  
  
  RESULTS
  This study shows that maximum SELP volumetric production is achieved at 37°C using terrific broth at pH 6-7.5, a shake flask volume to medium volume ratio of 10:1 and an agitation speed of 200 rpm. Maximum induction is attained at the beginning of the stationary phase with 0.5 mM IPTG and an induction period of at least 4 hours. We show that the selection agents ampicillin and carbenicillin are rapidly degraded early in the cultivation and that plasmid stability decreases dramatically on induction. Furthermore, acetate accumulates during the bioprocess to levels which are shown to be inhibitory to the host cells. Using our optimised conditions, 500 mg/L of purified SELP was obtained.
  
  
  CONCLUSIONS
  We have identified the optimal conditions for the shake flask production of a novel SELP with the final production levels obtained being the highest reported to date. While this study is focused on SELPs, we believe that it could also be of general interest to any study where the pET (ampicillin selective marker)-E. coli BL21(DE3) expression system is used. In particular, we show that induction time is critical in this system with, in contrast to that which is generally believed, optimal production being obtained by induction at the beginning of the stationary phase. Furthermore, we believe that we are at or near the maximum productivity for the system used, with rapid degradation of the selective agent by plasmid encoded β-lactamase, plasmid instability on induction and high acetate production levels being the principal limiting factors for further improved production.
authors: Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal. tcollins@bio.uminho.pt
authors: Tony
authors: T
authors: Collins
authors: João
authors: J
authors: Azevedo-Silva
authors: André
authors: A
authors: da Costa
authors: Fernando
authors: F
authors: Branca
authors: Raul
authors: R
authors: Machado
authors: Margarida
authors: M
authors: Casal
mesh_terms: D018533:Biomass; D004549:Elastin / Q000378:metabolism; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D047011:Silk / Q000378:metabolism; D013696:Temperature
chemical_list: D011506:Proteins; D011994:Recombinant Proteins; D047011:Silk; D004549:Elastin