Horseradish peroxidase (HRP) is an intensely studied enzyme with a wide range of commercial applications. Traditionally, HRP is extracted from plant; however, recombinant HRP (rHRP) production is a promising alternative. Here, non-glycosylated rHRP was produced in Escherichia coli as a DsbA fusion protein including a Dsb signal sequence for translocation to the periplasm and a His tag for purification. The missing N-glycosylation results in reduced catalytic activity and thermal stability, therefore enzyme engineering was used to improve these characteristics. The amino acids at four N-glycosylation sites, namely N13, N57, N255 and N268, were mutated by site-directed mutagenesis and combined to double, triple and quadruple enzyme variants. Subsequently, the rHRP fusion proteins were purified by immobilized metal affinity chromatography (IMAC) and biochemically characterized. We found that the quadruple mutant rHRP N13D/N57S/N255D/N268D showed 2-fold higher thermostability and 8-fold increased catalytic activity with 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as reducing substrate when compared to the non-mutated rHRP benchmark enzyme.

Inclusion bodies (IBs) are insoluble aggregates of misfolded protein in Escherichia coli. Against the outdated belief that the production of IBs should be avoided during recombinant protein production, quite a number of recombinant products are currently produced as IBs, which are then processed to give correctly folded and soluble product. However, this processing is quite cumbersome comprising IB wash, IB solubilization and refolding. To date, IB processing often happens rather uncontrolled and relies on empiricism rather than sound process understanding. In this mini review we describe current efforts to introduce more monitoring and control in IB processes, focusing on the refolding step, and thus generate process understanding and knowledge.

Horseradish peroxidase (HRP), an enzyme omnipresent in biotechnology, is still produced from hairy root cultures, although this procedure is time-consuming and only gives low yields. In addition, the plant-derived enzyme preparation consists of a variable mixture of isoenzymes with high batch-to-batch variation preventing its use in therapeutic applications. In this study, we present a novel and scalable recombinant HRP production process in Escherichia coli that yields a highly pure, active and homogeneous single isoenzyme. We successfully developed a multi-step inclusion body process giving a final yield of 960 mg active HRP/L culture medium with a purity of ≥99% determined by size-exclusion high-performance liquid chromatography (SEC-HPLC). The Reinheitszahl, as well as the activity with 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 3,3′,5,5′-tetramethylbenzidine (TMB) as reducing substrates, are comparable to commercially available plant HRP. Thus, our preparation of recombinant, unglycosylated HRP from E. coli is a viable alternative to the enzyme from plant and highly interesting for therapeutic applications.

Refolding is known as the bottleneck in inclusion body (IB) downstream processing in the pharmaceutical industry: high dilutions leading to large operating volumes, slow refolding kinetics and low refolding yields are only a few of the problems that impede industrial application. Solubilization prior to refolding is often carried out empirically and the effects of the solubilizate on the subsequent refolding step are rarely investigated. The results obtained in this study, however, indicate that the quality of the IB solubilizate has a severe effect on subsequent refolding. As the solubilizate contains chaotropic reagents in high molarities, it is commonly analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE, however, suffers from a long analysis time, making at-line analytical implementation difficult. In this study, we established an at-line reversed phase liquid chromatography method to investigate the time-dependent quality of the solubilizate. To verify the necessity of at-line solubilization monitoring, we varied the essential solubilization conditions for horseradish peroxidase IBs. The solubilization time was found to have a major influence on subsequent refolding, underlining the high need for an at-line analysis of solubilization. Furthermore, we used the developed reversed phase liquid chromatography method for an in-process control (IPC). In conclusion, the presented reversed phase liquid chromatography method allows a proper control of IB solubilization applicable for tailored refolding.