Functional expression of recombinant human stefin A in mammalian and bacterial cells

Abstract

Recombinant human cysteine protease inhibitor, stefin A, was expressed in both E. coli and BSC-1 monkey kidney cells utilizing pET and recombinant Vaccinia virus systems, respectively. The expressed protein was purified and analyzed by SDS-PAGE and western blot analysis utilizing a polyclonal antibody against rat cystatin α. In both cases the purified protein appeared as a single band corresponding to the molecular weight of stefin A (~10 kDa). Viability of the expressed stefin A was determined by the inhibition of the plant cysteine protease, papain. Recombinant human stefin A expressed in both E. coli and BSC-1 cells was shown to almost completely inhibit papain. The expression of a fully functional recombinant human stefin A in the bacterial system provides a highly efficient tool for the production of large quantities of the protein. This can be an important tool in kinetic studies as well as in production of antibodies for other analytical studies (immunoblot, immunohistochemical studies, etc.). Expression in the mammalian cells on the other hand, can provide a significant research tool to study the functional roles of stefin A in the mammalian systems such as the regulation of cysteine proteases.

Functional expression of amyloidogenic human stefins A and B in Pichia pastoris using codon optimization

“…The K i values are to be seen as approximate because corrections for substrate‐induced dissociation of enzyme–inhibitor complexes and formation of oligomeric states of stefins were not made . The K i value obtained was about six times higher than the value obtained using the E. coli expressed recombinant HSA and closely resembled the value obtained from the mutational study of E. coli expressed recombinant HSB . As previously reported, the differences in the value should not be overstressed because of the experimental difficulties of K i determination .…”

“…In the present study, we found that HSA and HSB exhibited different molecular stabilities during a 6‐day incubation at 37°C under neutral pH conditions, with HSB showing more vulnerability to fibril formation even under mild incubation conditions. Although the expression of functional recombinant HSA and HSB in E. coli has been previously reported , the proteins were contained N‐/C‐terminal extensions and/or were fused with tags. In addition, as recombinant HSA and HSB are often expressed as inclusion bodies in E. coli , an additional solubilizing process may be required for their purification.…”

Understanding Haemophilus parasuis infection in porcine spleen through a transcriptomics approach

“…Previous studies suggest that CSTA plays a critical role in the proliferation and differentiation of keratinocytes, and in regulating antigen presentation and apoptosis [ 66 - 68 ]. Importantly, down-regulation of CSTA has been detected in a wide range of epithelial tumors [ 69 ], which further indicates its important role in the differentiation and development of epithelial cells.…”