Koshiba T, Hayashi T, Miwako I, Kumagai I, Ikura T, Kawano K, Nitta K, Kuwajima K
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Kita-ku, Sapporo 060-0810, Japan.
Protein Eng. 1999 May;12(5):429-35. doi: 10.1093/protein/12.5.429.
A high-expression plasmid of the canine milk lysozyme, which belongs to the family of calcium-binding lysozymes, was constructed in order to study its physico-chemical properties. Because the cDNA sequence of the protein has not yet been determined, a 400 base-pair gene encoding canine milk lysozyme was first designed on the basis of the known amino acid sequence. The gene was constructed by an enzymatic assembly of 21 chemically synthesized oligonucleotides and inserted into an Escherichia coli expression vector by stepwise ligation. The expression plasmid thus constructed was transformed into BL21(DE3)/pLysS cells. The gene product accumulated as inclusion bodies in an insoluble fraction. Recombinant canine milk lysozyme was obtained by purification and refolding of the product and showed the same characteristics in terms of bacteriolytic activity and far- and near-UV circular dichroism spectra as the authentic protein. The NMR spectra of refolded lysozyme were also characteristic of a native globular protein. It was concluded that recombinant canine milk lysozyme was folded into the correct native structure. Moreover, the thermal unfolding profiles of the refolded recombinant lysozyme showed a stable equilibrium intermediate, indicating that the molten globule state of this protein was extraordinarily stable. This expression system of canine milk lysozyme will enable biophysical and structural studies of this protein to be extended.
为了研究犬乳溶菌酶的物理化学性质，构建了一种属于钙结合溶菌酶家族的犬乳溶菌酶高表达质粒。由于该蛋白质的cDNA序列尚未确定，首先根据已知的氨基酸序列设计了一个编码犬乳溶菌酶的400个碱基对的基因。该基因由21个化学合成的寡核苷酸通过酶促组装构建而成，并通过逐步连接插入到大肠杆菌表达载体中。将由此构建的表达质粒转化到BL21(DE3)/pLysS细胞中。基因产物以包涵体的形式积累在不溶性部分中。通过对产物进行纯化和重折叠获得重组犬乳溶菌酶，其在溶菌活性以及远紫外和近紫外圆二色光谱方面表现出与天然蛋白质相同的特性。重折叠溶菌酶的核磁共振光谱也具有天然球状蛋白质的特征。得出的结论是重组犬乳溶菌酶折叠成了正确的天然结构。此外，重折叠的重组溶菌酶的热解折叠曲线显示出一个稳定的平衡中间体，表明该蛋白质的熔融球状体状态异常稳定。这种犬乳溶菌酶表达系统将使对该蛋白质的生物物理和结构研究得以扩展。
