Antibody affinity maturation using yeast display with detergent-solubilized membrane proteins as antigen sources.
Antibody affinity maturation using yeast display with detergent-solubilized membrane proteins as antigen sources.
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使用酵母展示和去污剂溶解的膜蛋白作为抗原源进行抗体亲和力成熟。
DOI：
10.1093/protein/gzs077
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Shusta,EricV
中科院分区：
文献类型：
--
作者：
Tillotson,BenjaminJ;deLarrinoa,IñigoF;Skinner,ColinA;Klavas,DerekM;Shusta,EricV
原则上，去污剂溶解的细胞裂解物形式的抗原制剂可以使膜蛋白（MP）与体外抗体工程技术相容。为此，将去污剂溶解的细胞裂解物与酵母表面展示平台偶联以使抗转铁蛋白受体（TfR）单链抗体（scFv）亲和成熟。在经历质膜限制性生物素化后，通过用含去污剂的缓冲液溶解表达TfR的HEK 293细胞来产生裂解物。然后在竞争性解离速率筛选中将裂解物驻留的TfR与诱变性抗TfR scFv文库组合，并且鉴定了在酵母表面上具有高达4倍的提高的解离速率的scFv。重要的是，尽管裂解物含有生物素化蛋白质的复杂混合物，但工程改造的scFv保留了其TfR结合特异性。当作为可溶性蛋白由酵母分泌时，突变体scFv以3-7倍的平衡结合亲和力改善结合细胞表面TfR。尽管本研究针对的是已知的MP抗原，但采用生物素标记作为抗原检测手段使得基于裂解物的方法特别灵活。我们先前已经表明，酵母展示可以用于使用细胞裂解物驻留MP抗原来鉴定前导抗体，并且结合这项工作表明抗体也可以使用细胞裂解物定量工程化，这些方法可以提供用于产生和优化针对MP的抗体的高通量平台。
Antigen preparations in the form of detergent-solubilized cell lysates could, in principle, render membrane proteins (MPs) compatible within vitroantibody engineering technologies. To this end, detergent-solubilized cell lysates were coupled with the yeast surface display platform to affinity mature an anti-transferrin receptor (TfR) single-chain antibody (scFv). Lysates were generated from TfR-expressing HEK293 cells by solubilization with detergent-containing buffer after undergoing plasma membrane-restricted biotinylation. Lysate-resident TfR was then combined with a mutagenic anti-TfR scFv library in a competitive, dissociation rate screen, and scFvs were identified with up to 4-fold improved dissociation rates on the surface of yeast. Importantly, although the lysates contained a complex mixture of biotinylated proteins, the engineered scFvs retained their TfR binding specificity. When secreted by yeast as soluble proteins, mutant scFvs bound to cell surface TfR with 3–7-fold improvements in equilibrium binding affinity. Although a known MP antigen was targeted for purposes of this study, employing biotin tagging as a means of antigen detection makes the lysate-based approach particularly flexible. We have previously shown that yeast display can be used to identify lead antibodies using cell lysate-resident MP antigens, and combined with this work showing that antibodies can also be quantitatively engineered using cell lysates, these approaches may provide a high-throughput platform for generation and optimization of antibodies against MPs.
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DOI：
10.1073/pnas.95.11.6157
发表时间：
1998-05-26
影响因子：
11.1
作者：
Sheets, MD;Amersdorfer, P;Marks, JD
通讯作者：
Marks, JD
基于抗体的治疗方法的开发
DOI：
10.1007/978-1-4419-5955-3
发表时间：
2012
期刊：
Gastrointestinal and Liver Disorders in Women’s Health
影响因子：
--
作者：
M. Tabrizi;G. G. Bornstein;S. Klakamp
通讯作者：
S. Klakamp
影响因子：
5.6
作者：
Honegger, A;Plückthun, A
通讯作者：
Plückthun, A
定义细胞活化的人细胞表面抗原的单克隆抗体 (5E9) 的表征。
DOI：
--
发表时间：
1981
期刊：
Journal of immunology (Baltimore, Md. : 1950)
影响因子：
--
作者：
Haynes,BF;Hemler,M;Cotner,T;Mann,DL;Eisenbarth,GS;Strominger,JL;Fauci,AS
通讯作者：
Fauci,AS
影响因子：
5.6
作者：
Schier, R;Bye, J;Marks, JD
通讯作者：
Marks, JD