Efficient expression and purification of biologically active human cystatin proteins.
Cystatins are reversible cysteine protease inhibitor proteins. They are known to play important roles in controlling cathepsins, neurodegenerative disease, and in immune system regulation. Production of recombinant cystatin proteins is important for biochemical and function characterization. In this study, we cloned and expressed human stefin A, stefin B and cystatin C in Escherichia coli. Human stefin A, stefin B and cystatin C were purified from soluble fraction. For cystatin C, we used various chaperone plasmids to make cystatin C soluble, as it is reported to localize in inclusion bodies. Trigger factor, GroES-GroEL, DnaK-DnaJ-GrpE chaperones lead to the presence of cystatin C in the soluble fraction. Immobilized metal affinity chromatography, glutathione sepharose and anion exchange chromatography techniques were employed for efficient purification of these proteins. Their biological activities were tested by inhibition assays against cathepsin L and H3 protease.
Laboratory of Chromatin Biology, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, 462023, India.
Sakshi
S
Chauhan
Laboratory of Chromatin Biology, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, 462023, India. Electronic address: rst@iiserb.ac.in.
Raghuvir S
RS
Tomar
D056668:Cathepsin L / Q000037:antagonists & inhibitors / Q000737:chemistry; D055312:Cystatin A / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D055313:Cystatin B / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D055316:Cystatin C / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D055312:Cystatin A; D055316:Cystatin C; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D055313:Cystatin B; D010450:Endopeptidases; D056668:Cathepsin L; C021361:histone proteases
Cystatin C; DnaK-DnaJ-GrpE; GroES-GroEL; Stefin A; Stefin B; Trigger factor