Sianidis Giorgos, Pozidis Charalambos, Becker Fiona, Vrancken Kristof, Sjoeholm Carsten, Karamanou Spyridoula, Takamiya-Wik Monika, van Mellaert Lieve, Schaefer Thomas, Anné Jozef, Economou Anastassios
Institute of Molecular Biology and Biotechnology-FORTH and Department of Biology, University of Crete, P.O. Box 1527, Iraklio-Crete 71110, Greece.
J Biotechnol. 2006 Feb 24;121(4):498-507. doi: 10.1016/j.jbiotec.2005.08.002. Epub 2005 Sep 15.
The gene encoding a novel xyloglucanase (Xeg) belonging to family 74 glycoside hydrolases was isolated from a Jonesia sp. strain through functional screening in Escherichia coli. The encoded xyloglucanase is a protein of 972 aminoacyl residues with a 23 residue aminoterminal signal peptide. Over-expression of Xeg in B. subtilis or E. coli failed. In contrast, Xeg was successfully over-expressed and secreted in Streptomyces lividans TK24. To this end Xeg was fused C-terminally to the secretory signal peptide of the subtilisin inhibitor protein (vsi) from Streptomyces venezuelae. The native Xeg signal peptide derived from Jonesia sp. is only poorly functional in S. lividans. Under optimal growth conditions, significant amounts of mature Xeg (100-150 mg/l) are secreted in the spent growth media. A protocol to rapidly purify Xeg to homogeneity from culture supernatants was developed. Biophysical and biochemical analyses indicate that the enzyme is intact, stable and fully functional. Xeg is the longest heterologous polypeptide shown to be secreted from S. lividans. This study further validates use of S. lividans for production of active heterologous proteins and demonstrates that heterologous polypeptides of up to 100 kDa are also tractable by this system.
通过在大肠杆菌中进行功能筛选，从一种琼氏菌属菌株中分离出了编码属于74家族糖苷水解酶的新型木葡聚糖酶（Xeg）的基因。编码的木葡聚糖酶是一种含有972个氨酰基残基的蛋白质，带有一个23个残基的氨基末端信号肽。Xeg在枯草芽孢杆菌或大肠杆菌中未能成功过表达。相反，Xeg在变铅青链霉菌TK24中成功过表达并分泌。为此，将Xeg在C末端与来自委内瑞拉链霉菌的枯草杆菌蛋白酶抑制剂蛋白（vsi）的分泌信号肽融合。源自琼氏菌属的天然Xeg信号肽在变铅青链霉菌中的功能很差。在最佳生长条件下，大量成熟的Xeg（100 - 150 mg/l）分泌到用过的生长培养基中。开发了一种从培养上清液中快速纯化Xeg至同质的方案。生物物理和生化分析表明该酶完整、稳定且功能完全。Xeg是已证明从变铅青链霉菌分泌的最长的异源多肽。本研究进一步验证了使用变铅青链霉菌生产活性异源蛋白，并证明该系统也可处理高达100 kDa的异源多肽。
