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首页> 外文期刊>BMC Biotechnology >Improved mycobacterial protein production using a Mycobacterium smegmatis groEL1ΔC expression strain
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Improved mycobacterial protein production using a Mycobacterium smegmatis groEL1ΔC expression strain

机译：使用耻垢分枝杆菌groEL1ΔC表达菌株改善分枝杆菌蛋白质的生产

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Background The non-pathogenic bacterium Mycobacterium smegmatis is widely used as a near-native expression host for the purification of Mycobacterium tuberculosis proteins. Unfortunately, the Hsp60 chaperone GroEL1, which is relatively highly expressed, is often co-purified with polyhistidine-tagged recombinant proteins as a major contaminant when using this expression system. This is likely due to a histidine-rich C-terminus in GroEL1. Results In order to improve purification efficiency and yield of polyhistidine-tagged mycobacterial target proteins, we created a mutant version of GroEL1 by removing the coding sequence for the histidine-rich C-terminus, termed GroEL1ΔC. GroEL1ΔC, which is a functional protein, is no longer able to bind nickel affinity beads. Using a selection of challenging test proteins, we show that GroEL1ΔC is no longer present in protein samples purified from the groEL1ΔC expression strain and demonstrate the feasibility and advantages of purifying and characterising proteins produced using this strain. Conclusions This novel Mycobacterium smegmatis expression strain allows efficient expression and purification of mycobacterial proteins while concomitantly removing the troublesome contaminant GroEL1 and consequently increasing the speed and efficiency of protein purification.
机译：背景技术非致病性耻垢分枝杆菌广泛用作纯化结核分枝杆菌蛋白的近天然表达宿主。不幸的是，当使用此表达系统时，相对高表达的Hsp60分子伴侣GroEL1通常与多组氨酸标签的重组蛋白一起作为主要污染物被共纯化。这可能是由于GroEL1中富含组氨酸的C末端。结果为了提高多组氨酸标签的分枝杆菌靶蛋白的纯化效率和产量，我们通过去除富含组氨酸的C末端称为GroEL1ΔC的编码序列，创建了GroEL1的突变型。作为功​​能蛋白的GroEL1ΔC不再能够结合镍亲和力珠。使用一系列具有挑战性的测试蛋白质，我们显示从groEL1ΔC表达菌株纯化的蛋白质样品中不再存在GroEL1ΔC，并证明了纯化和表征使用该菌株生产的蛋白质的可行性和优势。结论该新型耻垢分枝杆菌表达菌株能够有效表达和纯化分枝杆菌蛋白，同时去除麻烦的污染物GroEL1，从而提高了蛋白纯化的速度和效率。

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