ABSTRACT
We describe here the expression of a C-terminally truncated form of human procollagenase-3 in Escherichia coli. The protein was found almost exclusively in inclusion bodies that were solubilized and refolded by two separate methods and then purified on Ni-NTA agarose. The purified proenzyme could be activated with either trypsin or APMA and active enzyme could be purified on a peptidic hydroxamate affinity column. Competitive elution from the affinity matrix yielded a highly purified preparation.
Subject(s)
Collagenases/metabolism , Escherichia coli/enzymology , Protein Folding , Chromatography, Affinity , Chromatography, Liquid , Collagenases/chemistry , Collagenases/genetics , Collagenases/isolation & purification , Electrophoresis, Polyacrylamide Gel , Enzyme Precursors/chemistry , Enzyme Precursors/genetics , Enzyme Precursors/isolation & purification , Enzyme Precursors/metabolism , Escherichia coli/genetics , Humans , Matrix Metalloproteinase 13ABSTRACT
Cell pastes and supernatant Escherichia coli samples, taken from an industrial bioprocess overproducing recombinant alpha 2 IFN were analysed using pyrolysis mass spectrometry (PyMS) and diffuse reflectance-absorbance Fourier transform infrared spectroscopy (FT-IR). PyMS and FT-IR are physico-chemical methods which measure predominantly the bond strengths of molecules and the vibrations of bonds within functional groups, respectively. They therefore give quantitative information about the total biochemical composition of the bioprocess sample. The interpretation of these hyperspectral data, in terms of the quantity of alpha 2 IFN in the cell pastes and supernatant samples was possible only after the application of the 'supervised learning' methods of artificial neural networks (ANNs) and partial least squares (PLS) regression. Both PyMS and FT-IR are novel, rapid and economical methods for the screening and the quantitative analysis of complex biological bioprocess over producing recombinant proteins. Models established using either spectral data set had a similarly satisfactory predictive ability. This shows that whole-reaction mixture spectral methods, which measure all molecules simultaneously, do contain enough information to allow their quantification when the entire spectra are used as the inputs to methods based on supervised learning. Moreover, this is the first study where FT-IR in the mid-IR range has been used to quantify the expression of a heterologous protein directly from fermentation broths and the first study to compare the abilities of PyMS and FT-IR for the quantitative analyses of an industrial bioprocess.
