Bolewska K, Kozłowska H, Goch G, Mikołajek B, Bierzyński A
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
Acta Biochim Pol. 1997;44(2):275-83.
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.
钙结合S100A1蛋白由两个S100α亚基组成。基于与其他S100蛋白的序列同源性，人们认为结合环是由S100α多肽链的19 - 32位和62 - 73位氨基酸残基形成的。在该蛋白的氧化形式中，亚基通过其Cys85残基之间的二硫键彼此共价连接。构建了编码牛S100α亚基的合成基因，并将其克隆到pAED4质粒的衍生物中。利用T7表达系统在大肠杆菌中表达该基因。通过质谱分析以及对其N端和C端进行测序对表达产物进行了纯化和鉴定。产生了三种不同形式（a、b和c）的S100α：具有天然序列、在N端带有起始甲硫氨酸、在C端带有额外的丙氨酸以及带有起始甲硫氨酸。该物质部分被氧化。有趣的是，仅形成了a、b和c种的同型二聚体。蛋白质的总产量约为50 mg/L培养物。通过定点诱变获得了编码S100α的Glu32→Gln和Glu73→Gln突变体的基因，并在同一系统中表达。在这两种情况下，都获得了氧化型和还原型a、b和c种的相似混合物。E73Q突变体的总产量与天然蛋白相似，而E32Q的总产量低约一半。正如预期的那样，S100α亚基的突变体仅结合一个钙离子。
