50L规模中试发酵重组核苷二磷酸激酶A工程菌

中试规模发酵重组人核苷二磷酸激酶A（rhNDPK-A）工程菌，并对表达产物进行纯化。摇瓶培养一级种子至合适密度，以10%比例接种二级种子培养基，在7L发酵罐中培养至OD600为9.6～10.5，然后转入80L发酵罐中进行补料分批培养，所得菌体裂解后，经离子交换层析和亲和层析两步纯化得重组蛋白制品。结果表明，50L培养液经过10h培养后，湿菌收量为1560 g/批，NDPK-A表达量为23.8%。另外，补料方式对发酵密度有明显影响。与单纯补加碳源相比，同时补加碳源和氮源可以显著提高菌体产量，但对目的蛋白表达量地提高不明显。在较优条件下，菌体产量为(2220.00±169.71) g/批，蛋白表达量为(22.00±0.42) %，纯化后重组蛋白得率为510mg/L。产物可溶、密度适中、工艺简便的中试发酵条件的建立为高得率、大规模制备重组rhNDPK-A奠定了基础。

Pilot-scale fermentation of rhNDPK-A producing E. coli in 50L culture

Production of recombinant human nucleoside diphosphate kinase A (rhNDPK-A) in pilot-scale. The primary seed culture was flask-shaked to 5.0~5.5 OD600, and then inoculated into 7L fermentor in ratio of 10%. Cultured to 9.6~10.5 OD600, the secondary seed culture was inoculated into 80L fermentor to carry out fed-batch culture. The obtained bacteria were homogenized under high-pressure and micro-filtered to remove the bacteria residue. Concentrated by superfilter, the crude rhNDPK-A was purified by ion-exchange and affinity chromatography. The results showed that the wet cell yield was 31.27g/L or 1560g/batch in 50L culture after 10h fermentation. The expression level of NDPK-A was 23.8%. In addition, the nutrition fed had significant effect on the density of culture. Compared with only carbon-material feeding, the density of culture was significantly enhanced in the condition of feeding carbon and nitrogen materials, but the expression level had not significant enhancement. In an optimum condition, the culture density was (38.30±0.28) OD600 U/mL; the wet cell yield was (2220.00±169.71)g/batch, i.e. (44.4±x3.4) g/L; the protein level was (22.00±0.42)%. Production of rhNDPK-A in pilot-scale provides materials for the basic research and new drug development of NDPK-A.