Institute of Microbiology, Technical University Braunschweig, Spielmannstrasse 7, 38106, Braunschweig, Germany.
Appl Microbiol Biotechnol. 2010 Mar;86(2):659-70. doi: 10.1007/s00253-009-2252-9. Epub 2009 Nov 12.
A set of different integrative expression vectors for the intracellular production of recombinant proteins with or without affinity tag in Aspergillus niger was developed. Target genes can be expressed under the control of the highly efficient, constitutive pkiA promoter or the novel sucrose-inducible promoter of the beta-fructofuranosidase (sucA) gene of A. niger in the presence or absence of alternative carbon sources. All expression plasmids contain an identical multiple cloning sequence that allows parallel construction of N- or C-terminally His6- and StrepII-tagged versions of the target proteins. Production of two heterologous model proteins, the green fluorescence protein and the Thermobifida fusca hydrolase, proved the functionality of the vector system. Efficient production and easy detection of the target proteins as well as their fast purification by a one-step affinity chromatography, using the His6- or StrepII-tag sequence, was demonstrated.
我们构建了一套适用于黑曲霉的整合表达载体，可以在细胞内表达带有或不带有亲和标签的重组蛋白。目的基因可以在高效、组成型的 pkiA 启动子或新型黑曲霉β-呋喃果糖苷酶（sucA）基因蔗糖诱导型启动子的控制下表达，无论是否存在替代碳源。所有表达质粒均含有相同的多克隆序列，可平行构建 N 端或 C 端带有 His6 和 StrepII 标签的目的蛋白。两种异源模型蛋白，绿色荧光蛋白和嗜热纤维梭菌水解酶的生产证明了该载体系统的功能。通过一步亲和层析，利用 His6 或 StrepII 标签序列，可高效生产、轻松检测目的蛋白，并快速纯化。
