Xu Xiaohua, Kashima Orie, Saito Akira, Azakami Hiroyuki, Kato Akio
Department of Biological Chemistry, Yamaguchi University, Yamaguchi, Japan.
Biosci Biotechnol Biochem. 2004 Jun;68(6):1273-8. doi: 10.1271/bbb.68.1273.
Two serine-rich heptapeptides, Ser-Ser-Ser-Lys-Ser-Ser-Ser (S6K) and Ser-Ser-Ser-Ser-Ser-Ser-Ser (S7), were fused to the C-terminus of chicken lysozyme (Lz) by genetic modification to improve the functional properties of lysozyme. The cDNAs of S6K-lysozyme (S6K-Lz) and S7-lysozyme (S7-Lz) were inserted into the expression vector of Pichia pastoris and secreted in yeast cultivation medium. The secretion amounts of S6K-Lz and S7-Lz were about 60% of that of wild-type lysozyme (Wt-Lz). The CD spectra showed that the conformation of S6K-Lz and S7-Lz was conserved regardless of the attachment of serine-rich peptides. The denaturation curves of S6K-Lz and S7-Lz also showed that the conformational changes were very small. The lytic activity of S6K-Lz and S7-Lz was almost the same as that of Wt-Lz, while the bactericidal activity against Escherichia coli of S6K-Lz and S7-Lz was greatly increased. The acetic acid-urea PAGE of phosphatase-treated S6K-Lz and S7-Lz indicated the possibility of phosphorylation of the fused serine-rich heptapeptides.
通过基因改造将两种富含丝氨酸的七肽，即丝氨酸-丝氨酸-丝氨酸-赖氨酸-丝氨酸-丝氨酸-丝氨酸（S6K）和丝氨酸-丝氨酸-丝氨酸-丝氨酸-丝氨酸-丝氨酸-丝氨酸（S7），融合到鸡溶菌酶（Lz）的C末端，以改善溶菌酶的功能特性。将S6K-溶菌酶（S6K-Lz）和S7-溶菌酶（S7-Lz）的cDNA插入毕赤酵母表达载体中，并在酵母培养基中分泌。S6K-Lz和S7-Lz的分泌量约为野生型溶菌酶（Wt-Lz）的60%。圆二色谱表明，无论富含丝氨酸的肽的连接情况如何，S6K-Lz和S7-Lz的构象都是保守的。S6K-Lz和S7-Lz的变性曲线也表明构象变化非常小。S6K-Lz和S7-Lz的溶菌活性与Wt-Lz几乎相同，而S6K-Lz和S7-Lz对大肠杆菌的杀菌活性大大提高。经磷酸酶处理的S6K-Lz和S7-Lz的醋酸-尿素聚丙烯酰胺凝胶电泳表明融合的富含丝氨酸的七肽有磷酸化的可能性。
