Preparation of active recombinant cathepsin K expressed in bacteria as inclusion body
Section snippets
Materials
The E. coli strain, JM109[recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 Δ(lac proAB)/F′(traD36 proAB lacIq lacZΔM15)], was used as a expression host. Expression vector with procathepsin K gene inserted into pQE-30 (Qiagen, USA) was a kind gift from Dr. Stephen Weiss in University of Michigan. E. coli culture medium was Luria–Bertani (LB; 1% Bacto peptone, 0.5% NaCl, and 0.5% yeast extract) containing ampicillin (). Prestained molecular weight markers were purchased from Gibco BRL
Expression, purification, and refolding of recombinant protein procathepsin K
Recombinant human procathepsin K was abundantly expressed in E. coli with N-terminal His-tag (Fig. 1). Recombinant protein expressed with IPTG induction showed approximate molecular weight of 38 kDa in SDS–PAGE gel, which corresponds to the size of procathepsin K. Recombinant procathepsin K was expressed as insoluble inclusion body and could be dissolved in denaturing buffer. This is advantageous in isolating recombinant insoluble procathepsin K from soluble proteins, since separation of
Acknowledgements
This work has been supported by Hannam University.
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