Efficient production of single-chain Fv antibody possessing rare codon linkers in fed-batch fermentation
Efficient production of single-chain Fv antibody possessing rare codon linkers in fed-batch fermentation
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DOI：
10.1016/j.jbiosc.2008.09.001
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发表时间：
2009-01-01
影响因子：
2.8
通讯作者：
Katoh, Shigeo
中科院分区：
文献类型：
--
作者：
Kumada, Yoichi;Sakan, Yoshinobu;Katoh, Shigeo
具有两种带或不带稀有密码子的多肽接头的单链Fv抗体（scFv），即scFv（G（4）S）（3）（R）和scFv No.10（带稀有密码子）以及scFv（G（4）S）（3）和scFv No.10（NR）（不带稀有密码子），在分批和补料分批发酵的可控条件下表达，以比较体积生产率和比生产率可溶形式的 scFv 水平。在分批发酵中，scFv (G(4)S)(3)(R)和scFv No.10，即具有稀有密码子连接子的scFv的体积生产率水平比具有不具有稀有密码子连接子的scFv高3-5倍。在指数补料系统控制的补料分批发酵中，转化子的细胞浓度以相似的比生长速率（0.1 h(-1)）增加，而具有稀有密码子连接体的scFv的比生产力水平比不具有稀有密码子连接体的scFv高1.6倍。这些结果表明，多肽接头基因中几个稀有密码子的存在增加了scFv的可溶量。这可能是由于多肽接头位置翻译速度暂时降低所致，从而为 V(H) 结构域的折叠提供了时间，并避免了未折叠的 V(H) 和 V(L) 结构域的氨基酸残基之间不利的相互作用。 scFv No. 10 和 scFv No. 10(NR) 比 scFv (G(4)S)(3)(R) 和 (G(4)S)(3) 更高的比生产力水平可能是由于基于氨基酸序列的多肽接头的稳定性差异造成的。因此，本研究中测试的稀有密码子连接体对于在补料分批或连续发酵中大规模生产可溶性和活性scFv非常有用，其中可以保持高细胞活性。 (C) 2008 年，日本生物技术协会。版权所有。
Single-chain Fv antibody (scFv) having 2 types of polypeptide linkers with or without rare codons, namely scFv (G(4)S)(3)(R) and scFv No.10 (with rare codons) and scFv (G(4)S)(3) and scFv No.10(NR) (without rare codon), were expressed under controllable conditions in batch and fed-batch fermentation, in order to compare volumetric productivity and specific productivity levels of scFvs as a soluble form. In batch fermentation, volumetric productivity levels of scFv (G(4)S)(3)(R) and scFv No.10, namely scFvs having the rare coclon linkers were 3-5 times higher than those of scFvs that had linkers without the rare codon. In fed-batch fermentation controlled by an exponential feeding system, the cell concentrations of the transformants increased with similar specific growth rates (0.1 h(-1)), while the specific productivity levels of scFvs with the rare codon linkers were 1.6 times higher than those of scFvs without the rare codon linkers. These results indicate that the presence of several rare codons in the gene of a polypeptide linker increases soluble amount of scFvs. This might be caused by a temporary decrease in translation speed at the position of the polypeptide linker allowing time for the folding of the V(H) domain and avoiding unfavorable interactions between amino acid residues at the unfolded V(H) and V(L) domains. Higher specific productivity levels of both scFv No. 10 and scFv No. 10(NR) than those of scFv (G(4)S)(3)(R) and (G(4)S)(3) might be caused by difference in stability of the polypeptide linkers on the basis of amino acid sequences. Thus, the rare codon linkers tested in this study will be considerably useful for large-scale production of soluble and active scFvs in fed-batch or continuous fermentations, in which high cell activity can be maintained. (C) 2008, The Society for Biotechnology, Japan. All rights reserved.