一个承载分子的设计与肽抗生素hPAB-β的高效表达

Design of a carrier molecule and high level expression of peptide antibiotics hPAB-β

目的 通过设计一个承载蛋白分子高效表达肽抗生素hPAB β ,为大量制备hPAB β奠定基础。 方法 根据肽抗生素的特性 ,确立 4条设计原则 ,并据此设计一 489bp的承载蛋白编码序列 ,用化学合成法获得该序列并将之克隆到pQE 3 2中构建成表达载体 ,进一步将肽抗生素hPAB β基因插入上述表达载体 ,肽抗生素基因位于承载蛋白编码序列 3’ 端 ,两者构成融合基因。将含融合基因的表达载体转化大肠杆菌JM10 9,筛选阳性重组子 ,并用阳性重组子表达融合蛋白。结果 设计的承载蛋白为 163个氨基酸 ,分子量 17668.80 ,等电点 5 .62 1。用承载分子与肽抗生素hPAB β构建的融合蛋白表达载体转化细菌后 ,筛选到 4个阳性重组子 ,均能高效表达肽抗生素融合蛋白 ,融合蛋白的产量占细菌总蛋白的 40 %～ 45 %。结论 设计的承载蛋白分子能够实现肽抗生素的高效表达。

Objective To design a carrier protein molecule which can express peptide antibiotics hPAB β at high level. Methods According to the properties of peptide antibiotics, a 489 bp DNA sequence coding for the carrier protein was designed under the help of DNAStar software. An expression plasmid pQE CH was constructed by inserting the fusion gene of designed DNA and peptide antibiotics hPAB β into pQE 32 plasmid. After the plasmid was transformed into E. coli JM109, 4 positive recombinants were selected and used to express the fusion proteins. Results All 4 selected recombinants containing the fusion gene of the designed carrier protein sequence and peptide antibiotics hPAB β expressed the fusion protein at high level about 40% to 45% of total cell proteins. Conclusion Expression plasmid pQE CH constructed from the designed carrier molecule and hPAB β expresses peptide antibiotics hPAB β at high level.