In vitro maturation pathway of a glutamyl endopeptidase precursor from Bacillus licheniformis.
A gene encoding of glutamyl-specific endopeptidase precursor from Bacillus licheniformis has been cloned in Escherichia coli cells. The recombinant protein was expressed and accumulated as cytoplasmic insoluble inclusion bodies. Washed inclusion bodies were solubilized in 6 M guanidine-HCL in the presence of reducing agent. The following precursor renaturation was performed by fast frequent dilution method. The highest yield of the refolded protein was achieved at pH value of 8.5 and 4 degrees C. The renaturation process was accompanied by a gradual splitting of Glu(-48)/Thr(-47) and Glu(-13)/Lys(-12) peptide bonds. A 26-kDa protein proved to be an end product of in vitro renaturation. The mature glutamyl endopeptidase with a molecular mass of 25 kDa was obtained after a limited proteolysis of the 26-kDa protein was performed by subtilisin or trypsin. The 26-kDa protein was purified by gel filtration on a Superdex 75 column. Comparative characteristics of the thermal stability and catalytic properties of the 26-kDa and 25-kDa proteins showed that complete cleavage of the N-terminal pro-peptide by exogenous proteinase is necessary for a final packing and activation of the B. licheniformis glutamyl endopeptidase.
Trachuk
L A
LA
Laboratory of Protein Chemistry, Institute of Genetics and Selection of Industrial Microorganisms, 1st Dorozhny, 1, Moscow, 113545, Russia. trachukl@mail.ru
Shcheglov
A S
AS
Milgotina
E I
EI
Chestukhina
G G
GG
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000235:genetics*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D020673:Protein Renaturation; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification
D004792:Enzyme Precursors; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase