Expression and characterization of an enantioselective antigen-binding fragment directed against α-amino acids.
Expression and characterization of an enantioselective antigen-binding fragment directed against α-amino acids.
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DOI：
10.1016/j.pep.2013.06.010
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发表时间：
2013-09
影响因子：
1.6
通讯作者：
Hofstetter O
中科院分区：
文献类型：
--
作者：
Eleniste PP;Hofstetter H;Hofstetter O
这项工作描述了立体选择性Fab的设计和表达，其具有与亲本单克隆抗体所显示的结合特性相当的结合特性。利用来自分泌立体选择性抗L-氨基酸抗体的杂交瘤克隆的mRNA，产生相应的生物技术产生的Fab。为此，基于广泛的文献和数据库检索设计适当的引物。用这些引物进行PCR扩增，成功地扩增了VH、VL、CL和CH 1基因片段。利用重叠PCR分别联合收割机VH和CH 1序列以及VL和CL序列，以获得编码HC和LC片段的基因。将这些序列分别克隆到pEXP 5-CT/TOPO表达载体中，并用于转染BL 21（DE 3）细胞。这两条链的单独表达，然后在重折叠缓冲液中组装，产生了Fab，其被证明与L-氨基酸结合，但不识别相应的D-对映体。
This work describes the design and expression of a stereoselective Fab that possesses binding properties comparable to those displayed by the parent monoclonal antibody. Utilizing mRNA from hybridoma clones that secrete a stereoselective anti-L-amino acid antibody, a corresponding biotechnologically produced Fab was generated. For that, appropriate primers were designed based on extensive literature and databank searches. Using these primers in PCR resulted in successful amplification of the VH, VL, CL and CH1 gene fragments. Overlap PCR was utilized to combine the VH and CH1 sequences and the VL and CL sequences, respectively, to obtain the genes encoding the HC and LC fragments. These sequences were separately cloned into the pEXP5-CT/TOPO expression vector and used for transfection of BL21(DE3) cells. Separate expression of the two chains, followed by assembly in a refolding buffer, yielded an Fab that was demonstrated to bind to L-amino acids but not to recognize the corresponding D-enantiomers.