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Use of a Derivative of Escherichia coli BL21(DE3)for Efficient Production of Three Different Recombinant Proteins

机译:大肠杆菌BL21(DE3)的衍生物用于高效生产三种不同重组蛋白的用途

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摘要

Plasmid instability and growth inhibition of plasmid-bearing cells after induction were encountered when E.coli BL21(DE3)was used as host for the production of antihuman ovarian carcinoma x antihuman CD3 single-chain bispecific antibody(AhOC x AhCD3),human soluble B lymphocyte stimulator fused with thioredoxin(Trx-hsBLyS)and human parathyroid hormone fused with thioredoxin(Trx-hPTH).A derivative of BL21(DE3),namely,BLRM(DE3),isolated and showing superiority in AhOC x AhCD3 production in our previous work,was further used here for more efficient production of these three different recombinant proteins.By using BLRM-(DE3)as host,the simplified one-stage fermentation process was developed,which was more labor-saving and yielded AhOC x AhCD3,comparable to that of the traditional two-stage fermentation process.Also,the plasmid stabilities and production yields of Trx-hsBLyS and Trx-hPTH were dramatically improved by the application of BLRM(DE3)instead of BL21-(DE3).A high Trx-hsBLyS yield(about 3.5 g/L)was obtained,which was more than twice as much as that of the recombinant BL21(DE3)strain.The Trx-hPTH yield was improved from about 700 mg/L to 1 g/L.These results further showed the superiority of BLRM(DE3)to BL21-(DE3)and suggested its effectiveness for other BL21(DE3)/pET heterologous protein expression systems,which encounter similar problems.
机译:当大肠杆菌BL21(DE3)用作生产抗人卵巢癌x抗人CD3单链双特异性抗体(AhOC x AhCD3),人可溶性B的宿主时,遇到诱导后质粒不稳定性和携带质粒的细胞的生长抑制硫氧还蛋白(Trx-hsBLyS)融合的淋巴细胞刺激物和硫氧还蛋白(Trx-hPTH)融合的人甲状旁腺激素。BL21(DE3)的衍生物,即BLRM(DE3),在我们以前的研究中分离并显示出AhOC x AhCD3产生的优越性通过将BLRM-(DE3)用作宿主,开发了简化的一阶段发酵工艺,该工艺更省力,并且可得到AhOC x AhCD3,可比Trx-hsBLyS和Trx-hPTH的质粒稳定性和产量通过使用BLRM(DE3)代替BL21-(DE3)而得到显着提高.Trx- hsBLyS产量(a结果得到3.5 g / L),是重组BL21(DE3)菌株的两倍多.Trx-hPTH产量从700 mg / L提高到1 g / L,这些结果进一步表明BLRM(DE3)相对于BL21-(DE3)的优越性,并提出了其对其他BL21(DE3)/ pET异源蛋白表达系统的有效性,这些系统也遇到类似的问题。

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