Recombinant pyruvate kinase type I from Escherichia coli: overproduction and revised C-terminus of the polypeptide.
The gene encoding pyruvate kinase type I (PKI) of Escherichia coli was amplified by PCR, cloned and sequenced. The gene product was overexpressed in E. coli, using an inducible T7 RNA polymerase-based expression system. The transformed cells contained sixtyfold the enzyme activity of the reference cells and enabled the purification of 30 mg of highly active PKI from 1 liter of culture. The gene sequence was determined and found to be different from the one previously reported, i.e., the T nucleotide at position 1351 was missing. This resulted in a downstream shift of the stop codon, thus the deduced polypeptide was 470 amino acids long instead of 462. In addition the twelve C-terminal amino acids of the former sequence were changed.
Valentini
G
G
Department of Biochemistry, University of Pavia, Italy.
Mattevi
A
A
Barilla
D
D
Galizzi
A
A
Speranza
M L
ML
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011770:Pyruvate Kinase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*
D001426:Bacterial Proteins; D011770:Pyruvate Kinase