人肽抗生素hPAB-β多拷贝串联基因工程菌的筛选及发酵研究

Screening of recombinant bacterium for expression of human peptide antibiotic hPAB-β multimers and evaluation of its fermentation

目的 :对已构建的人肽抗生素hPAB β 1～ 8拷贝串联基因工程菌进行筛选 ,并对其优势菌株进行发酵研究 ,为hPAB β的规模生产奠定基础。 方法 :对 1～ 8拷贝串联基因工程菌的目标蛋白表达率进行比较后 ,选择 2～ 5拷贝菌进行菌产量、目标蛋白表达率、包涵体溶解性、亲和层析效果比较 ,确定最佳多拷贝菌 ;对其摇瓶条件下发酵的培养液、温度、气体条件、IPTG诱导时机、浓度及时间进行研究。 结果 :在相同条件下 ,3拷贝菌产量(3.15 3g/L)最高 ,目标蛋白表达率 (2 7.8% )接近最高水平 ,包涵体能溶解完全 ;利用亲和层析能成功捕获融合蛋白 ,后者经羟胺裂解 ,可获得相对分子质量与合成hPAB β成熟肽大小相符的小肽 ,筛选出的优势菌传代稳定 ,其最佳摇瓶发酵条件为 37℃ ,16 0r/min条件下 ,在改良M 9 CAA培养液中培养至吸光值 (A60 0 )≈ 2 .5时 ,以终浓度为10 0 μmol/L的IPTG诱导表达 5h。 结论 :确定了 3拷贝菌为最佳多拷贝菌 ,并确定了其最佳摇瓶发酵参数。

Objective: To screen the best genetic engineering bacterium for the production of peptide antibiotic hPAB-β and evaluate its fermentation level in bottle. Methods:After analysis of the interest fusion protein expression levels of 8 recombinant bacteria containing 1-8 copies of human peptide antibiotic hPAB-β expressing plasmid respectively,2-5 copies expressing bacteria were chosen for the further study of their bacteria yield,expression forms of the target protein, dissolution of the inclusion bodies and the efficiency of fusion protein purification by affinity chromatography, then the best engineering bacterium with the certain copies of interest peptide expressing plasmid was screened out and its optimal fermentation parameters in bottle were also studied. Results:The recombinant bacterium transformed by 3 copies of interest peptide expressing plasmid was the best candidate for its bacteria yield (3.153 g/L) and fusion protein expression level (27.7%) were the highest among 1-8 copies candidates. The inclusion bodies of 3 copies target fusion protein could be easily dissolved by 8 mol/L urea and captured by Ni-NTA column. The elution of the fusion protein could be directly cleaved to monomer by adding 2 mol/L hydroxylamine, adjusting pH to 9.0 and incubating at 45℃ for 2 h. The optimal fermentation conditions of the selected recombinant bacteria were: culture the organisms with modified M9-CAA media at 37℃ and 160 r/min to (A 600 )≈2.5, then add IPTG to the final concentration 100 μmol/L to induce the expression of target fusion protein for 5 h. Conclusion:The engineering bacterium containing 3 copies interest peptide recombinant expressing plasmid is the best candidate for the production of peptide antibiotic hPAB-β,and its fermentation parameters are confirmed.