雷可肽生物合成中LxmX的表达纯化与结晶

Expression, Purification and Crystallization of LxmX in Lexapeptide Biosynthesis

雷可肽是一种对革兰氏阳性菌具有强抑制活性的第V类新型羊毛硫肽,其生物合成关键酶LxmX的蛋白晶体结构对雷可肽的生物合成机制解析至关重要。本研究构建了LxmX及其突变体LxmX’(S129/189M)的重组表达载体pPSUMO-lxmX和pPSUMO-lxmX’(S129/189M),并优化了表达条件,最终以Escherichia coli BL21(DE3)/pGro7为宿主,在0.1 mmol/L IPTG 30℃条件下诱导表达。通过镍柱亲和层析、TEV酶切、superdex200柱层析纯化得到纯度较高的蛋白。利用坐滴法点晶,20℃培养,经过结晶条件初筛和优化,得到了符合衍射条件的晶体。研究结果为解析LxmX的结构和研究雷可肽生物合成机制奠定了基础。

Lexapeptide is a novel class V lanthipeptide with strong inhibitory activity against gram-positive bacteria.The structure of the key enzymes,such as LxmX,is critical for the understanding of the biosynthesis mechanism of lexapeptide.In this study,the recombination vectors pPSUMO-lxmX and pPSUMO-lxmX’(S129/189M)respectively harboring the wild type and mutant LxmX were constructed.After optimization of the expression conditions,the proteins were successfully overexpressed with 0.1 mmol/L IPTG at 30℃in Escherichia coli BL21(DE3)/pGro7 and homogeneously purified through Ni2+column affinity chromatography.After removing sumo-tag by TEV protease digestion,LxmX and LxmX’(S129/189M)were concentrated by superdex200 column chromatography.The protein crystals were growing at 20℃using the sitting drop vapor diffusion method.After initial screening and optimization of crystallization conditions,we obtained crystals for diffraction.These results lay the foundation for resolving the LxmX atomic structure and elucidating its catalytic mechanism in lexapeptide biosynthesis.