Heterologous production of active ribonuclease inhibitor in Escherichia coli by redox state control and chaperonin coexpression

Abstract

BackgroundEukaryotic 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.ResultsRI 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).ConclusionsHigh 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.

Efficient E. coli Expression Strategies for Production of Soluble Human Crystallin ALDH3A1

“…Trigger factor, on the other hand, associates with the synthesized proteins as soon as they leave ribosome and through its interaction with their exposed hydrophobic patches averts their subsequent aggregation [14], [38]. Co-expression of molecular chaperones resulted in enhanced solubility and production of recombinant rice plant catalase A [39] active ribonuclease inhibitor [40], human scramblase 1 [41] and zeta-crystallin [42]. Solubility of his-tagged ALDH3A1 was significantly improved under conditions of co-expressing the pG-KJE8) suggesting that dnaK/dnaJ/grpE and groES/groEL are the essential chaperones for the correct folding of recombinant human ALDH3A1 (his-tagged ALDH3A1) when over-expressed in E. coli .…”

Consistent development of bioprocesses from microliter cultures to the industrial scale

“…Funke et al [37] equipped the BioLector instrument with a microfluidic control device to run fed-batch cultivations at microwell plate scale. This technology has been successfully applied in process developments for different recombinant proteins [22][23][24] and the robustness of the method in connection to scale-up to the 10 L scale was shown for a recombinant ribonuclease inhibitor byŠiurkus et al [25,39,40]. Three different liquids were pumped individually into each mini fermenter.…”

Increased Bacterial Cell Density and Recombinant Protein Yield Using a Commercial Microbial Cultivation System

“…Strategies range from simple media enrichment or modification (some examples include addition of reducing agents [7,8] or oxygen carriers [9] ) to the use of nutrient-releasing substrates that replicate aspects of fed-batch systems. EnBase (BioSilta) is a commercially available microbial cultivation system that increases cellular yield by utilizing enzymatically controlled release of glucose, from a polymeric substrate, to growth media.…”

“…[8] This study assessed the effect of EnBase media compared with Luria-Bertani (LB) media on attributes of bacterial cell yield and protein yield, solubility, and immunoreactivity of two recombinant capripoxvirus proteins.…”