An improved single-chain Fab platform for efficient display and recombinant expression.
An improved single-chain Fab platform for efficient display and recombinant expression.
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DOI：
10.1016/j.jmb.2014.11.017
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发表时间：
2015-01-30
影响因子：
5.6
通讯作者：
Wells, James A.
中科院分区：
文献类型：
--
作者：
Koerber, James T.;Hornsby, Michael J.;Wells, James A.
结合高通量选择的抗体噬菌体展示库最近显示出巨大的希望，可以创建下一代可再生的重组抗体，用于研究蛋白质及其许多翻译后修饰状态；然而，许多挑战仍然存在，如优化抗体支架。最近，一种单链Fab （scFab）格式，其中轻链的羧基端连接到重链的氨基端，被描述为潜在地结合了单链Fv的高显示水平和纯化Fab的高稳定性。然而，这种格式需要去除链间二硫键以达到适度的显示水平，随后的细菌表达导致高水平的scFab聚集，阻碍了scFab的进一步使用。在这里，我们开发了一种改进的scFab格式，通过增加轻链和重链之间的连接器长度来保留链间二硫键，以提高显示和细菌表达水平至每升1-3 mg。此外，将scFab重定向到共翻译信号识别颗粒（SRP）途径，并结合信号肽序列的重组，结果显示水平比原始scFab格式高24倍，比亲本Fab水平高3倍。这种优化的scFab支架可以很容易地在一个步骤中重组，在细菌或哺乳动物宿主中表达，以产生稳定（81°C Tm），主要是单体（>90%）的高产抗体。最终，这种新的scFab格式将推进高通量抗体生成平台，以发现下一代研究和治疗抗体。
Antibody phage display libraries combined with high-throughput selections have recently demonstrated tremendous promise to create the next generation of renewable, recombinant antibodies to study proteins and their many post-translational modification states; however many challenges still remain, such as optimized antibody scaffolds. Recently, a single-chain Fab (scFab) format, in which the carboxy-terminus of the light chain is linked to the amino-terminus of the heavy chain, was described to potentially combine the high display levels of a single-chain Fv with the high stability of purified Fabs. However, this format required removal of the interchain disulfide bond to achieve modest display levels and subsequent bacterial expression resulted in high levels of aggregated scFab, hindering further use of scFabs. Here, we developed an improved scFab format that retains the interchain disulfide bond by increasing the linker length between the light and heavy chains to improve display and bacterial expression levels to 1–3 mg per liter. Furthermore, rerouting of the scFab to the co-translational signal recognition particle (SRP) pathway combined with reengineering of the signal peptide sequence results in display levels 24-fold above the original scFab format and 3-fold above parent Fab levels. This optimized scFab scaffold can be easily reformatted in a single step for expression in a bacterial or mammalian host to produce stable (81°C Tm), predominantly monomeric (>90%) antibodies at a high yield. Ultimately, this new scFab format will advance high-throughput antibody generation platforms to discover the next generation of research and therapeutic antibodies.
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将噬菌体显示到高吞吐抗体的产生和表征。
影响因子：
12.3
作者：
Schofield, Darren J;Pope, Anthony R;Clementel, Veronica;Buckell, Jenny;Chapple, Susan Dj;Clarke, Kay F;Conquer, Jennie S;Crofts, Anna M;Crowther, Sandra R E;Dyson, Michael R;Flack, Gillian;Griffin, Gareth J;Hooks, Yvette;Howat, William J;Kolb-Kokocinski, Anja;Kunze, Susan;Martin, Cecile D;Maslen, Gareth L;Mitchell, Joanne N;O'Sullivan, Maureen;Perera, Rajika L;Roake, Wendy;Shadbolt, S Paul;Vincent, Karen J;Warford, Anthony;Wilson, Wendy E;Xie, Jane;Young, Joyce L;McCafferty, John
通讯作者：
McCafferty, John
影响因子：
14.9
作者：
Maupetit J;Derreumaux P;Tuffery P
通讯作者：
Tuffery P
影响因子：
2.4
作者：
Schlapschy, Martin;Grimm, Sebastian;Skerra, Arne
通讯作者：
Skerra, Arne
影响因子：
14.9
作者：
Baek, H;Suk, KH;Cha, S
通讯作者：
Cha, S
DOI：
10.1046/j.1432-1033.2002.03262.x
发表时间：
2002-11-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
Adams, H;Scotti, PA;Tommassen, J
通讯作者：
Tommassen, J