谷氨酸棒杆菌增强型表达载体构建及牛α-干扰素表达

Construction of an enhanced expression vector and expression of bovine interferon alpha using Corynebacterium glutamicum

谷氨酸棒杆菌是一种传统的食品级工业微生物,近年来正逐渐被开发成为一种新型的无内毒素重组蛋白表达宿主。为提高其外源蛋白的表达能力,将内源双顺反子元件引入到穿梭载体pXMJ19的多克隆位点之前,得到含有增强型Ptac启动子的谷氨酸棒杆菌表达载体pSM19,该载体以EGFP作为报告基因,相比出发载体表达量提高了99.5%。将密码子优化的,带有组氨酸标签的牛α-干扰素成熟蛋白编码基因合成并克隆到pSM19载体中,转化谷氨酸棒杆菌表达菌株Corynebacterium glutamicum CGMCC1.15647中进行诱导表达。培养温度为30℃时,重组牛α-干扰素蛋白大部分以包涵体形式存在,而16℃培养时的蛋白可溶性有较大改善。在5 L发酵罐中进行放大培养,得到大量表达重组牛α-干扰素的菌体,并用镍柱进行了简单纯化,产量预估为68 mg/L菌液。利用MDBK-VSV系统对重组牛α-干扰素进行生物学活性测定,其抗病毒效价为(1.35±0.23)×106 U/mg。

Corynebacterium glutamicum is a traditional food-grade industrial microorganism which has been developed as a novel endotoxin-free recombinant protein expression host in recent years. To improve the expressing ability of recombinant protein, an endogenous bicistronic element was introduced in front of the multiple cloning site of pXMJ19, bringing the plasmid an enhanced Ptac promoter. The new expression vector was named pSM19 and its expression strength was 99.5% higher than that of pXMJ19 measured by the enhanced green fluorescent protein reporter. The codon-optimized, histidine-tagged BoIFN-α encoding gene was then cloned into the pSM19 and the vector pSM19-BoIFN-α was transformed into the expression strain Corynebacterium glutamicum CGMCC1.15647. When cultured at 30 ℃, the recombinant BoIFN-α protein mostly existed in the form of inclusion bodies, while the solubility of which was greatly improved when cultured at 16 ℃. Scale-up culture was carried out in a 5 L bioreactor in order to obtain more cell pellets containing recombinant protein, followed by a simple purification procedure utilizing the nickel column. The yield of recombinant BoIFN-α was estimated at 68 mg/L and its antiviral activity was(1.35±0.23)×10~6 U/mg determined by the MDBK-VSV system.