The periplasmic Escherichia coli peptidylprolyl cis,trans-isomerase FkpA -: I.: Increased functional expression of antibody fragments with and without cis-prolines
The periplasmic Escherichia coli peptidylprolyl cis,trans-isomerase FkpA -: I.: Increased functional expression of antibody fragments with and without cis-prolines
复制标题
DOI：
10.1074/jbc.m910233199
复制
发表时间：
2000-06-02
影响因子：
4.8
通讯作者：
Plückthun, A
中科院分区：
文献类型：
--
作者：
Bothmann, H;Plückthun, A
大肠杆菌周质中重组蛋白的生产可能会因折叠问题而受限，导致周质聚集体的形成。我们使用了一种基于大肠杆菌文库与展示在丝状噬菌体上的低表达抗体单链Fv（scFv）片段共表达的周质伴侣蛋白选择系统（博特曼，H.，普吕克通，A.（1998年）《自然生物技术》16卷，376 - 380页）。通过对功能性抗体的筛选，之前已富集到蛋白质Skp，并表明它可提高多种scFv片段在周质中的表达。现在，用从一个skp缺陷型菌株的基因组DNA构建的文库重复这一选择策略，导致周质肽基脯氨酰顺反异构酶（PPIase）FkpA的富集。FkpA的共表达增加了噬菌体上展示的融合蛋白的量，并且极大地改善了即使是不含顺式脯氨酸的scFv片段在周质中的功能性表达。相比之下，周质PPIase PpiA和SurA的共表达并没有使周质中或噬菌体上展示的功能性scFv片段水平提高。结合随附论文中的体外数据（拉姆，K.，普吕克通，A.（2000年）《生物化学杂志》275卷，17106 - 17113页），我们得出结论：FkpA的作用与其PPIase活性无关。
The production of recombinant proteins in the periplasm of Escherichia coli can be limited by folding problems, leading to periplasmic aggregates. me used a selection system for periplasmic chaperones based on the coexpression of an E. coli library with a poorly expressing antibody single-chain Fv (scFv) fragment displayed on filamentous phage (Bothmann, H., and Pluckthun, A. (1998) Nature Biotechnol. 16, 376-380). By selection for a functional antibody, the protein Skp had been enriched previously and shown to improve periplasmic expression of a wide range of scFv fragments. This selection strategy was now repeated with a library constructed from the genomic DNA of an skp-deficient strain, leading to enrichment of the periplasmic peptidylprolyl cis,trans-isomerase (PPIase) FkpA. Coexpression of FkpA increased the amount of fusion protein displayed on the phage and dramatically improved functional periplasmic expression even of scFv fragments not containing cis-prolines. In contrast, the coexpression of the periplasmic PPIases PpiA and SurA showed no increase in the functional scFv fragment level in the periplasm or displayed on phage. Together with the in vitro data in the accompanying paper (Ramm, K., and Pluckthun, A. (2000) J. Biol. Chem. 275, 17106-17113), we conclude that the effect of FkpA is independent of its PPIase activity.