An X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis: cloning, expression in Escherichia coli, and application for removal of N-terminal Pro-Pro from recombinant proteins.
A novel pepX gene was cloned from isolated DNA of Lactococcus lactis by PCR. The deduced amino acid sequence of the 89-kDa protein showed 94, 93, 65, and 44% identity with the pepX protein from Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, Lactobacillus delbruecki subsp. bulgaricus, and Lactobacillus helveticus, respectively, and contained a serine protease G-K-S-Y-L-G consensus motif. The pepX gene has been cloned into pET17b and was expressed at a high level in Escherichia coli BL21 (DE3) LysS. PepX was purified to approximate homogeneity with ammonium sulfate precipitation and DEAE Sephadex A-50 chromatography. Optimal pepX activity was observed at pH 8.0 and 37 degrees C. According to SDS-PAGE analysis, pepX has a molecular mass of approximately 89 kDa. The peptidase can remove completely the unwanted X-Pro from the N-terminal of the target protein, releasing the naturally active protein and peptide, revealing a prospective application of pepX in large-scale production of pharmaceutical protein and peptide products.
Xin
Ming
M
Biopharmaceutical College, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, People's Republic of China.
Li
Yang
Y
Jie
Lin
L
Min
Ding
D
Liu
Jingjing
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D004151:Dipeptides / Q000378:metabolism; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism*; D016415:Sequence Alignment; D013696:Temperature
D004151:Dipeptides; D011994:Recombinant Proteins; C448039:prolyl-proline; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C098392:PepX dipeptidyl aminopeptidase