Heo Joo-Hyung, Won Hye Soon, Kang Hyun Ah, Rhee Sang-Ki, Chung Bong Hyun
Biomolecular Process Engineering Laboratory, Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejon 305-600, Korea.
Protein Expr Purif. 2002 Feb;24(1):117-22. doi: 10.1006/prep.2001.1527.
The gene encoding human epidermal growth factor (hEGF) was expressed as a fusion protein with the Saccharomyces cerevisiae-derived prepro alpha-factor leader in the methylotrophic yeast Hansenula polymorpha. The recombinant hEGF(1-53), when secreted by H. polymorpha, rapidly cleaved to hEGF(1-52) by carboxy-terminal proteolysis, resulting in the accumulation of C-terminal-truncated hEGF(1-52) in the culture medium. To solve this problem, we constructed a H. polymorpha mutant in which the KEX1 gene coding for carboxypeptidase ysc(alpha) was disrupted. The extent of C-terminal proteolysis of hEGF was significantly reduced when this kex1 disruptant was used as a host strain. After 24 h of shake-flask culture, most of the hEGF secreted by the kex1 disruptant remained intact, whereas more than 90% of the hEGF secreted by the wild-type was C-terminally cleaved. The recombinant hEGF was purified to >98% purity by two sequential steps of preparative scale anion exchange chromatography and reverse-phase HPLC. The authenticity of purified hEGF was confirmed by HPLC, N-terminal amino acid sequencing, and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analyses.
编码人表皮生长因子（hEGF）的基因在多形汉逊酵母中表达为与酿酒酵母来源的前原α因子前导序列融合的蛋白。多形汉逊酵母分泌的重组hEGF(1 - 53)通过羧基末端蛋白水解迅速裂解为hEGF(1 - 52)，导致培养基中积累了C末端截短的hEGF(1 - 52)。为了解决这个问题，我们构建了一个多形汉逊酵母突变体，其中编码羧肽酶ysc(α)的KEX1基因被破坏。当使用这个kex1破坏突变体作为宿主菌株时，hEGF的C末端蛋白水解程度显著降低。摇瓶培养24小时后，kex1破坏突变体分泌的大部分hEGF保持完整，而野生型分泌的hEGF超过90%发生了C末端裂解。通过两步制备规模的阴离子交换色谱和反相HPLC将重组hEGF纯化至纯度>98%。通过HPLC、N末端氨基酸测序和基质辅助激光解吸/电离飞行时间质谱分析证实了纯化的hEGF的真实性。
