Research Center for Healthy Breeding of Livestock and Poultry, Institute of Subtropical Agriculture, CAS, Changsha, Hunan 410125, China.
Appl Biochem Biotechnol. 2012 Feb;166(3):640-51. doi: 10.1007/s12010-011-9455-0. Epub 2011 Nov 23.
Bovine lactoferrampin (LFA) and bovine lactoferricin (LFC) are two antimicrobial peptides located in the N(1) domain of bovine lactoferrin. The bactericidal activity of the fused peptide LFA-LFC is stronger than that of either LFA or LFC. The high cost of peptide production from either native digestion or chemical synthesis limits the clinical application of antimicrobial peptides. The expression of recombinant peptides in yeast may be an effective alternative. In the current study, the expression, purification, and antibacterial activity of LFA-LFC using the Pichia pastoris expression system are reported. The linearized expression vector pPICZaA-LFA-LFC was transformed into P. pastoris KM71 by electroporation, and positive colonies harboring the target genes were screened out and used for fermentation. The recombinant LFA-LFC peptide was purified via two-step column chromatography and identified by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The results indicate that P. pastoris is a suitable system for secreting LFA-LFC. The fermentation supernate and the purified LFA-LFC show high antimicrobial activities. The current study is the first to report on the expression and purification of LFA-LFC in P. pastoris and may have potential practical applications in microbial peptide production.
牛乳铁传递蛋白（LFA）和牛乳铁蛋白肽（LFC）是两种位于牛乳铁蛋白 N（1）结构域的抗菌肽。融合肽 LFA-LFC 的杀菌活性强于 LFA 或 LFC。无论是从天然消化还是化学合成获得的肽的高成本限制了抗菌肽的临床应用。在酵母中表达重组肽可能是一种有效的替代方法。本研究报告了利用毕赤酵母表达系统表达、纯化和抗菌活性的 LFA-LFC。通过电穿孔将线性化的表达载体 pPICZaA-LFA-LFC 转化到毕赤酵母 KM71 中，筛选出含有靶基因的阳性菌落，并用于发酵。通过两步柱层析法纯化重组 LFA-LFC 肽，并通过三氯乙酸-十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和基质辅助激光解吸/电离飞行时间质谱进行鉴定。结果表明，毕赤酵母是分泌 LFA-LFC 的合适系统。发酵上清液和纯化的 LFA-LFC 显示出很高的抗菌活性。本研究首次报道了 LFA-LFC 在毕赤酵母中的表达和纯化，在微生物肽生产方面可能具有潜在的实际应用价值。
