title: Heterologous production of active ribonuclease inhibitor in Escherichia coli by redox state control and chaperonin coexpression.
abstract: BACKGROUND
  Eukaryotic Ribonuclease inhibitor (RI), belonging to the RNH1 family, is distinguished by unique features--a high sensitivity to oxidation due to the large number of reduced cysteins and a high hydrophobicity, which made most production approaches so far unsuccessful or resulted in very low yields. In this work efficient in vivo folding of native RI in the Escherichia coli cytoplasm was obtained by external addition of a reducing agent in tandem with oxygen limitation and overproduction of a molecular chaperonin. After optimisation of the production conditions in the shake flask scale the process was scaled up to high cell densities by applying a glucose limited fed-batch procedure.
  
  
  RESULTS
  RI production in a T7 RNA polymerase based system results in accumulation of aggregated inactive product in inclusion bodies. Combination of addition of the reductant DTT, low production temperature and coexpression of the chaperonin GroELS resulted in high level production of approximately 25 mg g(-1) CDW active RI in E. coli ER2566 pET21b, corresponding to approximately 800 kU g(-1) cell wet weight. Further conditional screening under fed-batch-like conditions with the EnBase® technology and scale up into the bioreactor scale resulted in an efficient high cell density glucose and oxygen limited fed-batch process with a final cell dry weight of 25 g L(-1) and a total RI yield of app. 625 mg L(-1) (volumetric activity of 80,000 kU L(-1)). The E. coli based production constructs showed a very high robustness. The recombinant culture maintained its productivity despite the combination of the toxic growth conditions, the substrate limited production mode in tandem with a high level expression of several recombinant proteins, the set of molecular chaperonins and the target protein (RI).
  
  
  CONCLUSIONS
  High level production of active RI in E. coli in a T7 RNA polymerase expression system depends on the following factors: (i) addition of a reducing agent, (ii) low production temperature, (iii) oxygen limitation, and (iii) co-overexpression of the chaperonin GroELS. The study indicates the strength of applying fed-batch cultivation techniques for the efficient optimisation of production factors already at the screening stage for fast and straight forward bioprocess development even for target proteins which show a complex folding behaviour. In our case none of the approaches alone would have resulted in significant accumulation of active RI.
authors.affiliation: Thermo Fisher Scientific, V.Graiciuno 8, LT-02241 Vilnius, Lithuania.
authors.forename: Juozas
authors.initials: J
authors.lastname: Šiurkus
authors.forename: Peter
authors.initials: P
authors.lastname: Neubauer
mesh_terms: D019149:Bioreactors; D018834:Chaperonin 60 / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D019163:Reducing Agents / Q000737:chemistry / Q000494:pharmacology; D012260:Ribonucleases / Q000037:antagonists & inhibitors; D013696:Temperature
chemical_list: D018834:Chaperonin 60; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D019163:Reducing Agents; D012260:Ribonucleases; D010100:Oxygen