Secretory production of single-chain antibody (scFv) in Brevibacillus choshinensis using novel fusion partner
Secretory production of single-chain antibody (scFv) in Brevibacillus choshinensis using novel fusion partner
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DOI：
10.1007/s00253-013-4695-2
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发表时间：
2013-10-01
影响因子：
5
通讯作者：
Arakawa, Tsutomu
中科院分区：
文献类型：
--
作者：
Tokunaga, Masao;Mizukami, Makoto;Arakawa, Tsutomu
嗜盐 β-内酰胺酶 (BLA) 已成功用作新型融合伴侣，用于在大肠杆菌细胞质中可溶性表达易于聚集的外源蛋白 (Appl Microbiol Biotechnol 86:649-658, 2010b)。这种嗜盐 BLA 融合技术在此应用于短芽孢杆菌的分泌表达。 Higeta Shoyu小组最近开发的“短芽孢杆菌体内克隆”方法用于短芽孢杆菌表达载体的构建和转化，有助于有效筛选短芽孢杆菌表达系统的生产条件。两种单链抗体（scFv），HyHEL-10单链scFv（scFvHEL）和抗荧光素单链scFv（scFvFLU），成功分泌到培养上清液中，作为与嗜盐BLA的融合蛋白。用凝血酶裂解 BLA 部分后纯化的 scFvHEL-His 具有完全活性：它与抗原、溶菌酶形成化学计量复合物，并抑制酶活性。 scFvFLU-His，以类似方式表达和纯化，化学计量地抑制荧光素的荧光强度。通过光散射测量确定 scFvHEL-His 的分子量为 27,800 Da，表明其在溶液中的单体结构。
Halophilic beta-lactamase (BLA) has been successfully used as a novel fusion partner for soluble expression of aggregation-prone foreign proteins in Escherichia coli cytoplasm (Appl Microbiol Biotechnol 86:649-658, 2010b). This halophilic BLA fusion technology was applied here for secretory expression in Brevibacillus. The "Brevibacillus in vivo cloning" method, recently developed by Higeta Shoyu group, for the construction and transformation of Brevibacillus expression vectors facilitates efficient screening of the production conditions of Brevibacillus expression system. Two single-chain antibodies (scFv), HyHEL-10 single chain scFv (scFvHEL) and anti-fluorescein single chain scFv (scFvFLU), were successfully secreted to culture supernatant as a fusion protein with halophilic BLA. The scFvHEL-His, purified after cleavage of BLA portion with thrombin, was fully active: it formed a stoichiometric complex with the antigen, lysozyme, and inhibited the enzymatic activity. The scFvFLU-His, similarly expressed and purified, stoichiometrically inhibited fluorescence intensity of fluorescein. The molecular mass of scFvHEL-His was determined to be 27,800 Da by light scattering measurements, indicating its monomeric structure in solution.