Production of Active Recombinant Hyaluronidase Inclusion Bodies from Apis mellifera in E. coli Bl21(DE3) and characterization by FT-IR Spectroscopy

Abstract

The bacterium E. coli is one of the most important hosts for recombinant protein production. The benefits are high growth rates, inexpensive media, and high protein titers. However, complex proteins with high molecular weight and many disulfide bonds are expressed as inclusion bodies (IBs). In the last decade, the overall perception of these IBs being not functional proteins changed, as enzyme activity was found within IBs. Several applications for direct use of IBs are already reported in literature. While fluorescent proteins or protein tags are used for determination of IB activity to date, direct measurements of IB protein activity are scacre. The expression of recombinant hyaluronidase from Apis mellifera in E. coli BL21(DE3) was analyzed using a face centered design of experiment approach. Hyaluronidase is a hard to express protein and imposes a high metabolic burden to the host. Conditions giving a high specific IB titer were found at 25 °C at low specific substrate uptake rates and induction times of 2 to 4 h. The protein activity of hyaluronidase IBs was verified using (Fourier transform) FT-IR spectroscopy. Degradation of the substrate hyaluronan occurred at increased rates with higher IB concentrations. Active recombinant hyaluronidase IBs can be immediately used for direct degradation of hyaluronan without further down streaming steps. FT-IR spectroscopy was introduced as a method for tracking IB activity and showed differences in degradation behavior of hyaluronan dependent on the applied active IB concentration.

Inhibition of E. coli Host RNA Polymerase Allows Efficient Extracellular Recombinant Protein Production by Enhancing Outer Membrane Leakiness

“…Thus, these process parameters are frequently investigated during upstream process development. [ 14,17–19 ] However, the contrary results in the aforementioned studies illustrate that the effects that temperature and μ have on OM leakiness might depend on a variety of factors, like the strain, product, or promoter, and are not fully understood yet.…”

Emerging Solutions for in Vivo Biocatalyst Immobilization: Tailor-Made Catalysts for Industrial Biocatalysis

“…Similarly, CatIBs containing the monomeric enzymes Amadoriase II ,,, and β-glycosidase from Thermus caldophilus as well as the industrially relevant enzyme lipase A from Bacillus subtilis ,,, have been produced. The clinically relevant, yet unstable and difficult to produce enzyme hyaluronidase from Apis mellifera was successfully generated as natural CatIBs (not requiring the fusion of a “pull-down” tag) in E. coli in a fed batch cultivation mode by the careful modification of the process parameters . The resulting hyaluronidase CatIBs were used to generate hyaluronan oligosaccharides, which can be employed to stimulate angiogenesis and tumor suppression.…”

“…The clinically relevant, yet unstable and difficult to produce enzyme hyaluronidase from Apis mellifera was successfully generated as natural CatIBs (not requiring the fusion of a "pull-down" tag) in E. coli in a fed batch cultivation mode by the careful modification of the process parameters. 76 The resulting hyaluronidase CatIBs were used to generate hyaluronan oligosaccharides, which can be employed to stimulate angiogenesis and tumor suppression.…”

Infrared Spectroscopy for Structure Analysis of Protein Inclusion Bodies

“…Due to the isolated position of the related absorption band and the high sensitivity to -sheet structures, IR spectroscopy is most suited for analysis of protein aggregates and IBs. Thus, IR spectroscopy was successfully employed to address different questions of IB analysis, including structure analysis [6][7][8][9][10][11], IB quantification [12], monitoring of IB formation kinetics [13], protein refolding [14] and IB activity [15].…”