Clinical manufacturing of recombinant human interleukin 15. I. Production cell line development and protein expression in E. coli with stop codon optimization.
Clinical manufacturing of recombinant human interleukin 15. I. Production cell line development and protein expression in E. coli with stop codon optimization.
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DOI：
10.1002/btpr.746
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发表时间：
2012-03
影响因子：
2.9
通讯作者：
Zhu J
中科院分区：
文献类型：
--
作者：
Vyas VV;Esposito D;Sumpter TL;Broadt TL;Hartley J;Knapp GC 4th;Cheng W;Jiang MS;Roach JM;Yang X;Giardina SL;Mitra G;Yovandich JL;Creekmore SP;Waldmann TA;Zhu J
白细胞介素 15 (IL-15) 在临床前模型中显示出促进 NK 细胞和 T 细胞活化和增殖以及增强 CD8+ T 细胞抗肿瘤免疫的显着生物学特性。在这里，我们报告了表达重组人白细胞介素 15 (rhIL-15) 的大肠杆菌细胞系的开发，用于临床生产。将人 IL-15 cDNA 序列插入 pET28b 质粒并在几种大肠杆菌 BL21 菌株中表达。通过对大肠杆菌BL21(DE3)、BL21(DE3)pLysS、BLR(DE3)pLysS、BL21-AI等多个大肠杆菌菌株的产品质量比较，选择大肠杆菌BL21-AI进行临床生产。在摇瓶和 20 L 发酵罐规模上进行表达优化，产物表达为包涵体，经过溶解、重折叠和纯化，产生活性 rhIL-15。根据肽图谱和质谱分析，15-20% 的纯化 rhIL-15 显示出对应于额外色氨酸残基的无关峰后，进一步研究了表达构建体的终止密码子。经确定，额外色氨酸的存在是由于终止密码子摆动效应所致，可以通过用 TAA（赭色）替换 TGA（蛋白石）终止密码子来消除该效应。作为一种新颖的策略，我们开发了一种简单的方法来证明生产宿主细胞中缺乏 tRNA 抑制因子，以验证本研究中的细胞。含有 rhIL-15 编码序列和三联体终止密码子 TAATAATGA 的大肠杆菌 BL21-AI 细胞被储存用于进一步的临床生产。
Interleukin 15 (IL-15) has shown remarkable biological properties of promoting NK- and T-cell activation and proliferation, as well as enhancing antitumor immunity of CD8+ T cells in preclinical models. Here, we report the development of an E. coli cell line to express recombinant human Interleukin-15 (rhIL-15) for clinical manufacturing. Human IL-15 cDNA sequence was inserted into a pET28b plasmid and expressed in several E. coli BL21 strains. Through product quality comparisons among several E. coli strains, including E. coli BL21(DE3), BL21(DE3)pLysS, BLR(DE3)pLysS, and BL21-AI, E. coli BL21-AI was selected for clinical manufacturing. Expression optimization was carried out at shake flask and 20-L fermenter scales, and the product was expressed as inclusion bodies that were solubilized, refolded, and purified to yield active rhIL-15. Stop codons of the expression construct were further investigated after 15–20% of the purified rhIL-15 showed an extraneous peak corresponding to an extra tryptophan residue based on peptide mapping and mass spectrometry analysis. It was determined that the presence of an extra tryptophan was due to a stop codon wobble effect, which could be eliminated by replacing TGA (opal) stop codon with TAA (ochre). As a novel strategy, a simple method of demonstrating lack of tRNA suppressors in the production host cells was developed to validate the cells in this study. The E. coli BL21-AI cells containing the rhIL-15 coding sequence with a triplet stop codon TAATAATGA were banked for further clinical manufacturing.
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影响因子：
14.9
作者：
BROWN, CM;STOCKWELL, PA;TATE, WP
通讯作者：
TATE, WP
影响因子：
56.9
作者：
GRABSTEIN, KH;EISENMAN, J;GIRI, JG
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GIRI, JG
影响因子：
4.1
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González, B;Ceciliani, F;Galizzi, A
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Galizzi, A
影响因子：
20.3
作者：
Lugli, Enrico;Goldman, Carolyn K.;Roederer, Mario
通讯作者：
Roederer, Mario
影响因子：
82.9
作者：
Teague, RM;Sather, BD;Greenberg, PD
通讯作者：
Greenberg, PD