摘要
目的 :利用原核重组表达技术 ,完成人可溶性肿瘤坏死因子相关凋亡诱导配体 (TRAIL ,又称凋亡素 2配体 ,Apo- 2 L )的中试。方法 :可溶性 TRAIL编码基因插入改建的 p BV 2 2 0载体 ;在发酵过程中 ,控制溶解氧在 30 %～ 5 0 % ,30℃生长 6 h,4 2℃诱导培养 4 h;菌体上清行金属亲和层析。结果 :发酵液中最终菌体密度 D(6 0 0 )达 8.0 (相当于每升发酵液含 15 g湿菌体 ) ,TRAIL占菌体总蛋白的 4 0 %左右 ,含量超过 0 .6 g/ L。其中 ,所表达的 TRAIL 2 0 %～ 30 %在上清而直接有活性 ,70 %～ 80 %呈包涵体。上清用亲和层析一步使其纯度达 70 %以上 ;包涵体超声破菌后经两次洗涤 ,纯度达到 80 %以上。 结论 :TRAIL
Objective:To prepare recom binant TRAIL by higher density cultivation of E.coli K80 2 / p BV- TRAIL in a3. 7L Bioengineering fermenter to overexpress recombinant soluble human TNF- related apoptosis- inducing ligand (TRAIL, Apo2 ligand,Apo2 L) .Methods:The gene of TRAIL code was inserted into rebuilt p BV2 2 0 .A feeding- in- batch process was used to limit glucose atlow level and keep dissolved oxygen at30 % - 5 0 % ;the bacteria grew at30℃ for6 h and raised to4 2℃ in 15 m in for4 h.TRAIL was isolated from the supernatant of bacteria by immobilized m etal affinity chrom atography (IMAC) .Results:The bacteria were reproduced in a large scale at the final cell density of D(6 0 0 ) =8(approximately15 g wet weight/ L) .Expressed TRAIL accounted for about4 0 % of total bacterial protein,which represented for0 .6 g/ L of final product.TRAIL presented as soluble form2 0 % - 30 % in supernatant and insoluble form 70 % - 80 % as inclusion bodies.The purity of soluble TRAIL was more than70 % after ultrasonication and IMAC,while the purity of inclusion bodies exceeded 80 % through ultrasonication and2 steps of washing.Conclusion:The recombinant TRAIL/ Apo2 L is highly expressed in E. coli K80 2 by higher density cultivation with feeding- in- batch protocol. [
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2002年第2期132-135,共4页
Academic Journal of Second Military Medical University
基金
国家自然科学基金资助项目 (30 0 0 0 0 78)
