Production and recovery of recombinant propapain with high yield.
Papain (EC 3.4.22.2), the archetypal cysteine protease of C1 family, is of considerable commercial significance. In order to obtain substantial quantities of active papain, the DNA coding for propapain, the papain precursor, has been cloned and expressed at a high level in Escherichia coli BL21(DE3) transformed with two T7 promoter based pET expression vectors - pET30 Ek/LIC and pET28a(+) each containing the propapain gene. In both cases, recombinant propapain was expressed as an insoluble His-tagged fusion protein, which was solubilized, and purified by nickel chelation affinity chromatography under denaturing conditions. By systematic variation of parameters influencing the folding, disulfide bond formation and prevention of aggregate formation, a straightforward refolding procedure, based on dilution method, has been designed. This refolded protein was subjected to size exclusion chromatography to remove impurities and around 400mg of properly refolded propapain was obtained from 1L of bacterial culture. The expressed protein was further verified by Western blot analysis by cross-reacting it with a polyclonal anti-papain antibody and the proteolytic activity was confirmed by gelatin SDS-PAGE. This refolded propapain could be converted to mature active papain by autocatalytic processing at low pH and the recombinant papain so obtained has a specific activity closely similar to the native papain. This is a simple and efficient expression and purification procedure to obtain a yield of active papain, which is the highest reported so far for any recombinant plant cysteine protease.
Choudhury
Debi
D
Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064, India.
Roy
Sumana
S
Chakrabarti
Chandana
C
Biswas
Sampa
S
Dattagupta
J K
JK
D029441:Carica / Q000201:enzymology*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D002479:Inclusion Bodies / Q000737:chemistry; D007700:Kinetics; D010206:Papain / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D004792:Enzyme Precursors; D011994:Recombinant Proteins; C071653:propapain; D010206:Papain