Expression of the copy DNA for human A4 and B4 L-lactate dehydrogenases in Escherichia coli.
The human LDH-A and LDH-B cDNAs, containing the coding regions for the L-lactate dehydrogenase A4 (M) and B4 (H) polypeptides respectively have been cloned into Escherichia coli to place the cDNAs under the control of hybrid E. coli/Bacillus stearothermophilus transcriptional and translational signals. Human A4- and B4-isoenzymes are produced in E. coli cells harbouring the expression plasmids pHLDHA22 and pHLDHB10 at levels of 6.5 and 1.5% of the soluble protein of the cell, respectively. The tac promoter of these vectors was not induced by isopropyl beta-D-thiogalactopyranoside. The A4 and B4 human isoenzymes synthesized in E. coli were purified to homogeneity and show the same properties as isoenzymes isolated from human tissue. The amino acid sequences of 12 N-terminal residues of the human isoenzymes synthesized in E. coli were determined to be identical to those deduced from the DNA sequence of the cloned cDNAs except that the N-terminal methionine was absent from both. However, in contrast to LDH made in human cells, acetylation of the N-terminal alanine does not take place in E. coli cells.
Barstow
D A
DA
Molecular Recognition Centre, University of Bristol School of Medical Sciences, U.K.
Black
G W
GW
Sharman
A F
AF
Scawen
M D
MD
Atkinson
T
T
Li
S S
SS
Chia
W N
WN
Clarke
A R
AR
Holbrook
J J
JJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis*; D004274:DNA, Recombinant / Q000096:biosynthesis; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics*; D007770:L-Lactate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids
D004274:DNA, Recombinant; D007527:Isoenzymes; D004247:DNA; D007770:L-Lactate Dehydrogenase; C037106:endodeoxyribonuclease NdeI; D015252:Deoxyribonucleases, Type II Site-Specific