Expression of Saccharomyces cerevisiae inorganic pyrophosphatase in Escherichia coli.
A segment of DNA encoding Saccharomyces cerevisiae inorganic pyrophosphatase (ppa gene) was amplified by the polymerase chain reaction. The pSCH1 and pSCB6 plasmids containing the ppa gene were obtained. Transformation of the E. coli BL21 strain with the resulting recombinant plasmids and selection of clones having extremely high expression of inorganic pyrophosphatase (PPase) were carried out. Superproduction of recombinant S. cerevisiae PPase up to 50% of the total bacterial protein was achieved. The enzyme was readily obtained and purified to homogeneity with the use of a simple purification technique. This work is the first description of S. cerevisiae PPase superproducer creation.
Kurilova
S A
SA
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russian Federation.
Vorobjeva
N N
NN
Nazarova
T I
TI
Avaeva
S M
SM
D001483:Base Sequence; D017931:DNA Primers; D004271:DNA, Fungal / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005800:Genes, Fungal; D043564:Inorganic Pyrophosphatase; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011755:Pyrophosphatases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D017931:DNA Primers; D004271:DNA, Fungal; D011994:Recombinant Proteins; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase