Recombinant pyruvate kinase type I from Escherichia coli: Overproduction and revised C-terminus of the polypeptide

G  Valentini; A  Mattevi; D  Barilla; A  Galizzi; M L  Speranza

Recombinant pyruvate kinase type I from Escherichia coli: Overproduction and revised C-terminus of the polypeptide

G Valentini, A Mattevi, D Barilla, A Galizzi, M L Speranza

Biology

Research output: Contribution to journal › Article › peer-review

Abstract

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.

Original language	English
Pages (from-to)	719-721
Number of pages	3
Journal	Journal of Biological Chemistry
Volume	378
Issue number	7
Publication status	Published - Jul 1997

Keywords

cloning
overexpression
sequence
SITES
DNA

Cite this

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title = "Recombinant pyruvate kinase type I from Escherichia coli: Overproduction and revised C-terminus of the polypeptide",

abstract = "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.",

keywords = "cloning, overexpression, sequence, SITES, DNA",

author = "G Valentini and A Mattevi and D Barilla and A Galizzi and Speranza, {M L}",

year = "1997",

month = jul,

language = "English",

volume = "378",

pages = "719--721",

journal = "Journal of Biological Chemistry",

issn = "0021-9258",

publisher = "American Society for Biochemistry and Molecular Biology Inc.",

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TY - JOUR

T1 - Recombinant pyruvate kinase type I from Escherichia coli: Overproduction and revised C-terminus of the polypeptide

AU - Valentini, G

AU - Mattevi, A

AU - Barilla, D

AU - Galizzi, A

AU - Speranza, M L

PY - 1997/7

Y1 - 1997/7

N2 - 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.

AB - 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.

KW - cloning

KW - overexpression

KW - sequence

KW - SITES

KW - DNA

M3 - Article

SN - 0021-9258

VL - 378

SP - 719

EP - 721

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 7

ER -