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首页> 外文期刊>Protein Expression and Purification >Expression, purification, and isotope labeling of the Fv of the human HIV-1 neutralizing antibody 447-52D for NMR studies
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Expression, purification, and isotope labeling of the Fv of the human HIV-1 neutralizing antibody 447-52D for NMR studies

机译：人类HIV-1中和抗体447-52D Fv的表达，纯化和同位素标记，用于NMR研究

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The Fv is the smallest antigen binding fragment of the antibody and is made of the variable domains of the light and heavy chains, V-L and V-H, respectively. The 26-kDa Fv is amenable for structure determination in solution using multi-dimensional hetero-nuclear NMR spectroscopy. The human monoclonal antibody 447-52D neutralizes a broad spectrum of HIV-1 isolates. This anti-HIV-1 antibody elicited in an infected patient is directed against the third variable loop (W) of the envelope glycoprotein (gp120) of the virus. The V3 loop is an immunodominant neutralizing epitope of HIV-1. To obtain the 447-52D Fv for NMR studies, an Escherichia coli bicistronic expression vector for the heterodimeric 447-52D Fv and vectors for single chain Fv and individually expressed V-H and V-L were constructed. A pelB signal peptide was linked to the antibody genes to enable secretion of the expressed polypeptides into the periplasm. For easy cloning of any antibody gene without potential modification of the antibody sequence, restriction sites were introduced in the pelB sequence and following the termination codon. A set of oligonucleotides that prime the leader peptide genes of all potential antibody human antibodies were designed as backward primers. The forward primers for the V-L and V-H were based on constant region sequences. The 447-52D Fv could not be expressed either by a bicistronic vector or as single chain Fv, probably due to its toxicity to Escherichia coli. High level of expression was obtained by individual expression of the VH and the VL chains, which were then purified and recombined to generate a soluble and active 447-52D Fv fragment. The V-L of mAb 447-52D was uniformly labeled with C-13 and N-15 nuclei (U-C-13/N-15). Preliminary NMR spectra demonstrate that structure determination of the recombinant 447-52D Fv and its complex with V3 peptides is feasible. (C) 2003 Elsevier Science (USA). All rights reserved. [References: 52]
机译：Fv是抗体的最小抗原结合片段，由轻链和重链的可变域（分别为V-L和V-H）组成。 26 kDa Fv适用于使用多维异核NMR光谱法测定溶液中的结构。人单克隆抗体447-52D中和了多种HIV-1分离株。在感染患者中引起的这种抗HIV-1抗体针对该病毒的包膜糖蛋白（gp120）的第三个可变环（W）。 V3环是HIV-1的免疫优势中和表位。为了获得用于NMR研究的447-52D Fv，构建了用于异二聚体447-52D Fv的大肠杆菌双顺反子表达载体以及用于单链Fv和单独表达的V-H和V-L的载体。 pelB信号肽连接到抗体基因，以使表达的多肽分泌到周质中。为了容易克隆任何抗体基因而又不对抗体序列进行可能的修饰，将限制性位点引入到pelB序列中并在终止密码子之后。将引发所有潜在抗体人抗体的前导肽基因的一组寡核苷酸设计为反向引物。 V-L和V-H的正向引物基于恒定区序列。 447-52D Fv不能通过双顺反子载体或单链Fv来表达，可能是由于其对大肠杆菌的毒性。通过单独表达VH和VL链可获得高水平的表达，然后将其纯化并重组以生成可溶且有活性的447-52D Fv片段。 mAb 447-52D的V-L均匀标记有C-13和N-15核（U-C-13 / N-15）。初步的NMR谱图表明重组447-52D Fv及其与V3肽的复合物的结构测定是可行的。 （C）2003 Elsevier Science（美国）。版权所有。 [参考：52]

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