Optimized Production of Active α-Glucosidase by Recombinant Escherichia coli. Evaluation of Processes Using in Vivo Reactivation from Inclusion Bodies

Abstract

During fast production in recombinant Escherichia coli, the enzyme alpha-glucosidase from Saccharomyces cerevisiae accumulates partially as inclusion bodies. The inclusion bodies are reactivated inside the cell upon temperature downshift. This in vivo reactivation was most efficient on complex medium with inclusion body production at 42 degrees C and reactivation at 30 degrees C, yielding volumetric activities 85% higher than those of extended isothermal production at low temperature. On defined medium, however, in vivo reactivation was slow. In fed-batch cultivations, feeding controls the specific growth rate independent of the temperature. Here, high growth rates fostered inclusion body formation even at low temperature. Intermediate growth rates permitted accumulation of active alpha-glucosidase without affecting the total amount of alpha-glucosidase. Low growth rates yielded similar activities and additionally prevented inclusion body formation. Moreover, high growth rates during production forestalled subsequent in vivo reactivation. Accumulation of activity after temperature reduction was possible with intermediate growth rates. The best time for temperature shift was concomitant to induction. Thus, in fed-batch culture, isothermal production at 30 degrees C and with a set growth rate of 0.12 h(-)(1) controlled by feeding was most efficient for production of active alpha-glucosidase. Compared to production under optimal conditions on complex medium, the specific and volumetric activities obtained were 3 and 45 times higher, respectively.

Enhanced growth and recombinant protein production of Escherichia coli by a perfluorinated oxygen carrier in miniaturized fed-batch cultures

“…This may be due to the higher turnover of inclusion bodies as earlier described e.g. for α-glucosidase [27], which is dependent on ATP. This observation is interesting and will be further investigated.…”

Manufacturing of recombinant therapeutic proteins in microbial systems

“…Therefore, IB paste or suspension contains reasonably pure protein aggregates, which can be stored frozen for at least several months, thus providing flexibility for production plants due to uncoupling fermentation and purification processes. Whether a product remains soluble or aggregates in the cytoplasm depends on intrinsic properties of the peptide or protein sequence, but also on promoter strength and fermentation parameters such as temperature and growth rate (for review see [35]). Choosing an appropriate fusion partner may influence the solubility and proteolytic stability.…”

Decreasing translation error rate in Escherichia coli increases protein function

“…2 ). This suggests that alpha-glucosidase activity can be increased without changing the growth conditions as done in [ 4 ]. However, by combining these two approaches it may be possible to obtain alpha-glucosidase with superior activity.…”

“…This can be done either by optimizing the purification processes, or by optimizing the microorganisms used to overexpress the protein. For example, lowering the growth temperature and the medium pH can help correct folding and improve the activity of alpha-glucosidase expressed in E. coli [ 4 ]. Overexpressing various protein chaperons in E. coli (GroEL/GroES, DnaK/DnaJ/GrpE, ClpB and the small HSPs lbpA/IbpB), alongside desired protein overexpression, has been shown to increase the yield of soluble protein [ 5 ].…”

“…As new genes are expressed and screened for protein activity, in order to make their production financially viable, the ability to express proteins in sufficient amount and quality at a reasonable cost becomes a priority [ 2 ]. Since it meets the affordability criteria and is easily manipulated and grown on large scale, E. coli is the most widely used prokaryotic system for the synthesis of heterologous proteins, including alpha-glucosidase and beta-glucanase [ 4 , 5 , 9 – 11 ]. We reasoned that reducing the error rate during translation via rpsL141 mutation, thus reducing proteome susceptibility to oxidative modification, would not only be beneficial for the entire proteome, but would directly and indirectly improve the quality of individual proteins and improve their specific activity.…”