Codon optimization, expression, and characterization of an internalizing anti-ErbB2 single-chain antibody in Pichia pastoris
Codon optimization, expression, and characterization of an internalizing anti-ErbB2 single-chain antibody in Pichia pastoris
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DOI：
10.1016/j.pep.2005.11.014
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发表时间：
2006-05-01
影响因子：
1.6
通讯作者：
Liu, Jing
中科院分区：
文献类型：
--
作者：
Hu, Siyi;Li, Liangwei;Liu, Jing
抗ErbB 2抗体被用作探索ErbB 2功能机制和治疗ErbB 2过表达肿瘤的方便工具。当我们利用毕赤酵母表达抗ErbB 2单链抗体（scFv）时，在摇瓶培养中，产量不超过1-2 mg/L。考虑到密码子使用偏好性差可能是导致翻译和scFv生产效率低下的一个限制因素，我们通过选择巴斯德毕赤酵母偏好的密码子，同时保持G + C含量在相对低的水平，设计并合成了全长scFv基因。密码子优化使scFv表达水平增加3至5倍，并高达6-10 mg/L。北方印迹进一步证实了scFv表达量的增加主要是由于翻译效率的提高。培养条件研究表明，甲醇诱导72小时后，在pH 6.5-7.0、2%酪蛋白氨基酸的条件下，细胞生长和单链抗体表达达到最大。分泌的scFv通过使用Ni 2+螯合亲和层析在一个步骤中容易地从培养上清液纯化（> 95%均质产物）。产率约为10-15 mg/L。功能研究表明，A21 scFv可以在与ErbB 2过表达细胞结合后高效内化，这表明该试剂可能被证明对ErbB 2靶向免疫治疗特别有用。(c)2005年爱思唯尔公司All rights reserved.
Anti-ErbB2 antibodies are used as convenient tools in exploration of ErbB2 functional mechanisms and in treatment of ErbB2-overexpressing tumors. When we employed the yeast Pichia pastoris to express an anti-ErbB2 single-chain antibody (scFv) derived from the tumor-inhibitory monoclonal antibody A21, the yield did not exceed 1-2 mg/L in shake flask cultures. As we considered that the poor codon usage bias may be one limiting factor leading to the inefficient translation and scFv production, we designed and synthesized the full-length scFv gene by choosing the P. pastoris preferred codons while keeping the G + C content at relatively low level. Codon optimization increased the scFv expression level 3- to 5-fold and up to 6-10 mg/L. Northern blotting further confirmed that the increase of scFv expression was mainly due to the enhancement of translation efficiency. Investigation of culture conditions revealed that the maximal cell growth and scFv expression were achieved at pH 6.5-7.0 with 2% casamino acids after 72 h methanol induction. Secreted scFv was easily purified (> 95% homogeneous product) from culture supernatants in one step by using Ni2+ chelating affinity chromatography. The yield was approximately 10-15 mg/L. Functional studies showed that the A21 scFv could be internalized with high efficiency after binding to the ErbB2-overexpressing cells, suggesting this regent may prove especially useful for ErbB2-targeted immunotherapy. (c) 2005 Elsevier Inc. All rights reserved.