In vitro refolding of carboxypeptidase T precursor from Thermoactinomyces vulgaris obtained in Escherichia coli as cytoplasmic inclusion bodies.
Carboxypeptidase T precursor from Thermoactinomyces vulgaris, which fails to contain its own leader peptide, has been expressed in Escherichia coli as insoluble cytoplasmic inclusion bodies. The yield of a washed recombinant protein from 1 L of culture liquid was about 60 mg. The obtained inclusion bodies were denatured in 6 M guanidine-HCl and then renatured by a rapid dilution. The important role of calcium for the complete stabilization of the refolded carboxypeptidase T precursor was established. After removal of minor admixture proteins by gel-filtration through Superdex 75, an electrophoretically homogeneous preparation of the native precursor of carboxypeptidase T was obtained. Processing of the resulting protein by subtilisin led to the formation of the mature carboxypeptidase T in which N-terminal sequence, molecular size, thermal stability, and catalytic properties were comparable to those of the natural enzyme.
Trachuk
Lesya
L
Laboratory of Protein Chemistry, Institute of Genetics and Selection of Industrial Microorganisms, 1st Dorozhny, 1, Moscow 113545, Russia. trachuk1@mail.ru
Letarov
Andrei
A
Kudelina
Irina A
IA
Yusupova
Margarita P
MP
Chestukhina
Galina G
GG
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002118:Calcium / Q000502:physiology; D002268:Carboxypeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000378:metabolism; D008848:Micromonosporaceae / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D011498:Protein Precursors; D011994:Recombinant Proteins; D002268:Carboxypeptidases; C076614:CPT protein, Thermoactinomyces vulgaris; D002118:Calcium