Brockmeier Ulf, Wendorff Marion, Eggert Thorsten
Institut für Molekulare Enzymtechnologie, Heinrich-Heine Universität Düsseldorf, Forschungszentrum Jülich, D-52426, Jülich, Germany.
Curr Microbiol. 2006 Feb;52(2):143-8. doi: 10.1007/s00284-005-0231-7. Epub 2006 Jan 31.
Most expression systems are based on Escherichia coli as the host strain because of the large availability of all kinds of vector plasmids. However, aside from the obvious advantages of E. coli systems, serious problems can occur during the process of heterologous gene expression and purification. Therefore, low expression rates, formation of inclusion bodies, improper protein-folding, and/or toxicity problems might enforce changing the expression host. Here we describe the construction of two new vectors, pBSMuL1 and pBSMuL2, for overexpression and secretion of heterologous proteins in Bacillus subtilis as an alternative expression host. The new plasmids combine several advantages in comparison to available Bacillus expression systems: an appropriate multiple cloning site consisting of 13 unique restriction sites, one (pBSMuL1) or two (pBSMuL2) strong constitutive promoters, a high efficient signal sequence for protein secretion, and the possibility to express proteins as His-tagged fusions for easy detection and purification. We have demonstrated the applicability of the novel vector plasmids for the production and purification of the heterologous cutinase from Fusarium solani pisi.
由于各种载体质粒的大量可得性，大多数表达系统都以大肠杆菌作为宿主菌株。然而，除了大肠杆菌系统的明显优势外，在异源基因表达和纯化过程中可能会出现严重问题。因此，低表达率、包涵体形成、蛋白质折叠不当和/或毒性问题可能会促使更换表达宿主。在此，我们描述了两种新载体pBSMuL1和pBSMuL2的构建，用于在枯草芽孢杆菌中过表达和分泌异源蛋白质，作为替代表达宿主。与现有的芽孢杆菌表达系统相比，新质粒具有几个优点：一个由13个独特限制酶切位点组成的合适多克隆位点、一个（pBSMuL1）或两个（pBSMuL2）强组成型启动子、一个用于蛋白质分泌的高效信号序列，以及将蛋白质表达为His标签融合蛋白以便于检测和纯化的可能性。我们已经证明了新型载体质粒在生产和纯化来自豌豆镰刀菌的异源角质酶方面的适用性。
