Max Planck Institute of Biophysics, Max-von-Laue Str. 3, 60438, Frankfurt am Main, Germany.
Microb Cell Fact. 2017 Sep 20;16(1):157. doi: 10.1186/s12934-017-0771-0.
Studies on membrane proteins are often hampered by insufficient yields of the protein of interest. Several prokaryotic hosts have been tested for their applicability as production platform but still Escherichia coli by far is the one most commonly used. Nevertheless, it has been demonstrated that in some cases hosts other than E. coli are more appropriate for certain target proteins.
Here we have developed an expression system for the heterologous production of membrane proteins using a single plasmid-based approach. The gammaproteobacterium Pseudomonas stutzeri was employed as a new production host. We investigated several basic microbiological features crucial for its handling in the laboratory. The organism belonging to bio-safety level one is a close relative of the human pathogen Pseudomonas aeruginosa. Pseudomonas stutzeri is comparable to E. coli regarding its growth and cultivation conditions. Several effective antibiotics were identified and a protocol for plasmid transformation was established. We present a workflow including cloning of the target proteins, small-scale screening for the best production conditions and finally large-scale production in the milligram range. The GFP folding assay was used for the rapid analysis of protein folding states. In summary, out of 36 heterologous target proteins, 20 were produced at high yields. Additionally, eight transporters derived from P. aeruginosa could be obtained with high yields. Upscaling of protein production and purification of a Gluconate:H Symporter (GntP) family transporter (STM2913) from Salmonella enterica to high purity was demonstrated.
Pseudomonas stutzeri is an alternative production host for membrane proteins with success rates comparable to E. coli. However, some proteins were produced with high yields in P. stutzeri but not in E. coli and vice versa. Therefore, P. stutzeri extends the spectrum of useful production hosts for membrane proteins and increases the success rate for highly produced proteins. Using the new pL2020 vector no additional cloning is required to test both hosts in parallel.
膜蛋白的研究经常受到目的蛋白产量不足的阻碍。已经测试了几种原核宿主作为生产平台，但迄今为止，大肠杆菌仍然是最常用的一种。然而，已经证明在某些情况下，宿主除了大肠杆菌以外，对于某些目标蛋白更合适。
本文开发了一种使用基于质粒的单一方法异源生产膜蛋白的表达系统。采用γ变形菌恶臭假单胞菌作为新的生产宿主。我们研究了几个对其在实验室中处理至关重要的基本微生物学特征。该生物属于生物安全一级，是人类病原体铜绿假单胞菌的近亲。恶臭假单胞菌在生长和培养条件方面与大肠杆菌相当。鉴定了几种有效的抗生素，并建立了质粒转化的方案。我们提出了一个包括目标蛋白克隆、最佳生产条件的小规模筛选以及最终在毫克范围内大规模生产的工作流程。GFP 折叠测定法用于快速分析蛋白质折叠状态。总之，在 36 种异源靶蛋白中，有 20 种得到了高产量的表达。此外，还可以从铜绿假单胞菌获得八种高产量的转运蛋白。展示了从沙门氏菌中高纯度规模化生产和纯化葡萄糖酸盐：H 同向转运体（GntP）家族转运体（STM2913）的蛋白。
恶臭假单胞菌是一种替代的膜蛋白生产宿主，其成功率与大肠杆菌相当。然而，有些蛋白在恶臭假单胞菌中得到了高产量的表达，但在大肠杆菌中却没有，反之亦然。因此，恶臭假单胞菌扩展了膜蛋白有用的生产宿主范围，并增加了高产量蛋白的成功率。使用新的 pL2020 载体，无需额外克隆即可同时测试两种宿主。
