Chen Hsiao-Ling, Lai Yi-Wen, Yen Chih-Ching, Lin Yuh-Yih, Lu Chien-Yu, Yang Shang-Hsun, Tsai Tung-Chou, Lin Yi-Jiun, Lin Chin-Wen, Chen Chuan-Mu
Department of Molecular Biotechnology, Da-Yeh University, Changhwa, Taiwan.
J Mol Microbiol Biotechnol. 2004;8(3):141-9. doi: 10.1159/000085787.
Lactoferrin is a metal-binding glycoprotein exhibiting multifunctional immunoregulation of antibacterial, antioxidant, anti-endotoxin and antiviral activities. Uptake of porcine lactoferrin (PLF) has been shown to enhance resistance to diarrhea and anemia in neonatal piglets. In this study, the methylotrophic yeast, Pichia pastoris, was used to express a recombinant PLF (rPLF) gene from swine mammary gland. A synthetic secretion cassette was constructed using the inducible promoter of the alcohol oxidase-1 gene (AOX1) and the yeast alpha-mating factor signal peptide. After electroporation and Zeocin selection, several clones expressed high levels of rPLF protein which constitutes more than 30% of the total protein. A time-course study showed that rPLF mRNA transcripts are stably expressed during 120 h of culture induction. rPLF was exported into the culture supernatant at approximately 87 mg/l and a large portion of rPLF was accumulated in the cell cytoplasm at approximately 760 mg/l after 72 h of methanol induction. Recombinant PLF protein was purified via a heparin column using a fast protein liquid chromatography system. The glycosylation of P. pastoris-derived rPLF was analyzed and similar patterns to milk PLF were observed. Pepsin hydrolysate of rPLF displayed high bactericidal activity against Escherichia coli ATCC 25922 under scanning electron microscopy observation and minimal inhibitory concentration and minimal bactericidal concentration tests. Our results suggested that the methylotrophic yeast-inducible system is suitable for large-scale production of active antibacterial rPLF glycoprotein.
乳铁蛋白是一种金属结合糖蛋白，具有抗菌、抗氧化、抗内毒素和抗病毒等多种免疫调节功能。研究表明，仔猪摄取猪乳铁蛋白（PLF）可增强其对腹泻和贫血的抵抗力。在本研究中，利用甲基营养酵母毕赤酵母表达来自猪乳腺的重组PLF（rPLF）基因。使用醇氧化酶-1基因（AOX1）的诱导型启动子和酵母α-交配因子信号肽构建了一个合成分泌盒。经电穿孔和博来霉素筛选后，多个克隆表达了高水平的rPLF蛋白，其占总蛋白的比例超过30%。一项时间进程研究表明，在120小时的培养诱导过程中，rPLF mRNA转录本稳定表达。甲醇诱导72小时后，rPLF以约87 mg/l的浓度分泌到培养上清液中，并且约760 mg/l的rPLF大量积累在细胞质中。使用快速蛋白质液相色谱系统通过肝素柱对重组PLF蛋白进行纯化。分析了毕赤酵母来源的rPLF的糖基化情况，并观察到其与乳PLF的糖基化模式相似。在扫描电子显微镜观察以及最低抑菌浓度和最低杀菌浓度测试中，rPLF的胃蛋白酶水解产物对大肠杆菌ATCC 25922显示出高杀菌活性。我们的结果表明，甲基营养酵母诱导系统适用于大规模生产具有活性抗菌作用的rPLF糖蛋白。
