Functional expression of recombinant human stefin A in mammalian and bacterial cells.
Recombinant human cysteine protease inhibitor, stefin A, was expressed in both Escherichia coli and BS-C-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 alpha. In both cases the purified protein appeared as a single band corresponding to the molecular weight of stefin A ( approximately 10kDa). 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 BS-C-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 mammalian systems such as regulation of cysteine proteases.
Calkins
Catharine C
CC
Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield, Detroit, MI 48201, USA.
Dosescu
Julie
J
Day
Nancy A
NA
Ren
Wei-Ping
WP
Fridman
Rafael
R
Sloane
Bonnie F
BF
Moin
Kamiar
K
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D055313:Cystatin B; D015891:Cystatins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D000882:Haplorhini; D006801:Humans; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid
C524994:CSTB protein, human; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D011994:Recombinant Proteins; D055313:Cystatin B