Section for Evolution and Genetics, Department of Biosciences, University of Oslo, Oslo, Norway.
Interfaculty Institute for Biochemistry, Eberhard Karls University, Tübingen, Germany.
Front Cell Infect Microbiol. 2017 Nov 7;7:464. doi: 10.3389/fcimb.2017.00464. eCollection 2017.
Almost all integral membrane proteins found in the outer membranes of Gram-negative bacteria belong to the transmembrane β-barrel family. These proteins are not only important for nutrient uptake and homeostasis, but are also involved in such processes as adhesion, protein secretion, biofilm formation, and virulence. As surface exposed molecules, outer membrane β-barrel proteins are also potential drug and vaccine targets. High production levels of heterologously expressed proteins are desirable for biochemical and especially structural studies, but over-expression and subsequent purification of membrane proteins, including outer membrane proteins, can be challenging. Here, we present a set of deletion mutants derived from BL21 Gold (DE3) designed for the over-expression of recombinant outer membrane proteins. These strains harbor deletions of four genes encoding abundant β-barrel proteins in the outer membrane (OmpA, OmpC, OmpF, and LamB), both single and in all combinations of double, triple, and quadruple knock-outs. The sequences encoding these outer membrane proteins were deleted completely, leaving only a minimal scar sequence, thus preventing the possibility of genetic reversion. Expression tests in the quadruple mutant strain with four test proteins, including a small outer membrane β-barrel protein and variants thereof as well as two virulence-related autotransporters, showed significantly improved expression and better quality of the produced proteins over the parent strain. Differences in growth behavior and aggregation in the presence of high salt were observed, but these phenomena did not negatively influence the expression in the quadruple mutant strain when handled as we recommend. The strains produced in this study can be used for outer membrane protein production and purification, but are also uniquely useful for labeling experiments for biophysical measurements in the native membrane environment.
几乎所有在革兰氏阴性细菌外膜中发现的整合膜蛋白都属于跨膜β桶家族。这些蛋白质不仅对营养物质的摄取和体内平衡很重要，而且还参与诸如粘附、蛋白质分泌、生物膜形成和毒力等过程。作为表面暴露的分子，外膜β桶蛋白也是潜在的药物和疫苗靶标。对于生化研究，特别是结构研究，异源表达蛋白的高产量是理想的，但膜蛋白（包括外膜蛋白）的过表达和随后的纯化可能具有挑战性。在这里，我们介绍了一组源自 BL21 Gold (DE3) 的缺失突变体，旨在过表达重组外膜蛋白。这些菌株缺失了编码外膜中丰富的β桶蛋白（OmpA、OmpC、OmpF 和 LamB）的四个基因，包括单基因缺失和双基因、三基因和四基因缺失的所有组合。这些外膜蛋白的编码序列被完全缺失，只留下一个最小的疤痕序列，从而防止了遗传回复的可能性。用四个测试蛋白（包括一个小的外膜β桶蛋白及其变体以及两种与毒力相关的自转运蛋白）在四重突变体菌株中的表达测试表明，与亲本菌株相比，表达量显著提高，且产生的蛋白质质量更好。在高盐存在下观察到生长行为和聚集的差异，但当按照我们的建议处理时，这些现象并没有对四重突变体菌株中的表达产生负面影响。本研究中产生的菌株可用于外膜蛋白的生产和纯化，但对于在天然膜环境中进行生物物理测量的标记实验也具有独特的用途。
