Molecular cloning and expression in Escherichia coli of a gene coding for bovine S100A1 protein and its Glu32-->Gln and Glu73-->Gln mutants.
Calcium binding S100A1 protein consists of two S100 alpha subunits. On the basis of sequence homology to other S100 proteins it is believed that the binding loops are formed by amino-acid residues 19-32 and 62-73 of S100 alpha polypeptide chain. In the oxidized form of the protein the subunits are linked covalently with each other by a disulphide bond between their Cys85 residues. A synthetic gene coding for bovine S100 alpha subunit was constructed and cloned into a derivative of pAED4 plasmid. The gene was expressed in Escherichia coli utilizing the T7 expression system. The expression products were purified and identified using mass spectrometry and by sequencing of their N- and C-termini. Three different forms (a, b, and c) of S100 alpha were produced: with the native sequence, with the initiator methionine at the N-terminus, and with an additional alanine at the C-terminus as well as with the initiator methionine. The material was partly oxidized. Interestingly, only the homodimers of a, b, and c species were formed. The total yield of the protein was about 50 mg/l of culture. Genes coding for Glu32-->Gln and Glu73-->Gln mutants of S100 alpha were obtained by site-directed mutagenesis and expressed in the same system. In both cases similar mixtures of oxidized and reduced a, b, and c species have been obtained. The total yield of E73Q mutant is similar to that of the native protein and that of E32Q lower by about a half. As expected, the mutants of S100 alpha subunits bind only one calcium ion.
Bolewska
K
K
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
Kozłowska
H
H
Goch
G
G
Mikołajek
B
B
Bierzyński
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D005973:Glutamine / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009418:S100 Proteins; D013056:Spectrophotometry, Ultraviolet
D002135:Calcium-Binding Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D009418:S100 Proteins; C110294:S100A1 protein; D005973:Glutamine; D018698:Glutamic Acid