关键词: 谷氨酸棒杆菌, 启动子, 蛋白表达, 牛α-干扰素

Abstract: 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)×106 U/mg determined by the MDBK-VSV system.

Key words: Corynebacterium glutamicum, promoter, protein expression, bovine interferon-α