Xiong Runsong, Chen Jingjing, Chen Jinchun
College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, People's Republic of China.
Biotechnol Appl Biochem. 2008 Nov;51(Pt 3):129-34. doi: 10.1042/BA20070205.
hLM (human lysozyme) has important potential application as a future safely administered human drug and food additive. To produce secreted rhLM (recombinant hLM) from the yeast Pichia pastoris, the signal peptide from HSA (human serum albumin) was employed to direct secreted expression. On the basis of the vector pPIC3.5k, an overexpression vector, pPIC3.5k-hLM, carrying the strong promoter AOX1 (aldehyde oxidase 1), the HSA signal peptide, the enterokinase recognition motif, the lysozyme gene and other necessary genetic segments was constructed and this was followed by a series of genetic manipulations. A positive colony was picked off to test its expression pattern. The target protein, rhLM, was obtained from the supernatant and showed a gradual enrichment with the induction time course, reaching its highest level at 72 h. This pattern was identical with that shown by the secreted expression of a heterologous protein directed by Saccharomcyes cerevisiae a-mating factor prepro-signal peptide in P. pastoris. After a series of purification processes, including ultrafiltration with a hollow-fibre membrane module, DEAE-Sepharose, Sephadex G50 chromatography and enterokinase digestion, the mature protein was characterized by MALDI-TOF-MS/MS (matrix-assisted laser-desorption ionization-time-of-flight tandem MS), N-terminal amino acid sequencing, and K(m) and K(cat) determination. The results confirmed that the rhLM was identical with native hLM. Moreover, the mature protein exhibited in vitro bacteriolytic activity against the Gram-positive bacterium Micrococcus lysodeikticus and the Gram-negative bacterium Escherichia coli. Taken together, it appeared that the HSA signal peptide was able direct secretive expression of a heterologous protein in P. pastoris.
人溶菌酶（hLM）作为一种未来可安全使用的人类药物和食品添加剂具有重要的潜在应用价值。为了利用毕赤酵母生产分泌型重组人溶菌酶（rhLM），采用了人血清白蛋白（HSA）的信号肽来指导分泌表达。基于载体pPIC3.5k构建了一个携带强启动子AOX1（醛氧化酶1）、HSA信号肽、肠激酶识别基序、溶菌酶基因和其他必要基因片段的过表达载体pPIC3.5k-hLM，随后进行了一系列基因操作。挑取一个阳性菌落来检测其表达模式。目标蛋白rhLM从培养上清中获得，并随着诱导时间的延长而逐渐富集，在72小时时达到最高水平。这种模式与酿酒酵母α-交配因子前体信号肽在毕赤酵母中指导异源蛋白分泌表达的模式相同。经过一系列纯化过程，包括中空纤维膜组件超滤、DEAE-琼脂糖、Sephadex G50层析和肠激酶消化，通过基质辅助激光解吸电离飞行时间串联质谱（MALDI-TOF-MS/MS）、N端氨基酸测序以及米氏常数（Km）和催化常数（Kcat）测定对成熟蛋白进行了表征。结果证实rhLM与天然hLM相同。此外，成熟蛋白对革兰氏阳性菌溶壁微球菌和革兰氏阴性菌大肠杆菌表现出体外溶菌活性。综上所述，HSA信号肽似乎能够在毕赤酵母中指导异源蛋白的分泌表达。
