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The cloning, characterisation and engineering of an IGF-I-BINDING single chain Fv

机译:IGF-I-BINDING单链Fv的克隆,鉴定和工程改造

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摘要

This thesis describes the construction and characterisation of an insulin-like growth factor (IGF-I)-binding single chain Fv (scFv) and the utilisation of this scFv as a model protein for the study of the application of DNA shuffling and ribosome display to antibody engineering. The variable domain genes were isolated from the hybridoma cell line producing the monoclonalantibody and successfully joined by PCR for the construction of the scFv, named anti-GPE.Sequencing of the gene revealed an unusually short heavy chain CDR2 region. The cloned scFv was expressed in E. coli and purified. Expression levels were low and the protein has poor solubility, most likely due to a reduction in folding efficiency caused by the abbreviated CDR2. The purified monomeric form of the protein was analysed for binding to IGF-I using surface plasmon resonance on the BIAcore 1000 with the specificity of the IgG version of the antibody for the three N-terminal residues of IGF-I - Gly-Pro-Glu - reproduced. The scFv's calculated dissociation constant of 3.68 µM is a low affinity for an antibody and is approximately 36-fold weaker than was calculated for the Fabversion of the antibody, but it is concluded that the calculated affinity for the scFv was an apparent affinity that may be an underestimation of true affinity due to the presence of non-functional or misfolded scFv species within the gel-filtration purified monomer peaks. A mutant version of anti-GPE with residues inserted in the CDR2 to restore it to normal length produced a protein with improved expression and solubility characteristics while retaining IGF-I-binding. It was concluded that the shortCDR2 was due to deletions generated during the somatic mutation process and a model for this isdescribed.A ribosome display method using a rabbit reticulocyte lysate as a source of ribosomes was developed for specific selection of anti-GPE against IGF-I. Error prone PCR was used to produce a random point mutated library of anti-GPE (EPGPE). This was taken through several cycles of display and selection but selection for non-specifically binding scFvs was commonly observed. This was probably due to poor folding of ribosome-displayed proteins in the system used, possibly caused by the presence of DTT in the lysate and/or the low capacity of the anti-GPE framework to tolerate mutation while retaining stability. It is assumed misfolds, exposing hydrophobic regions, would have a tendency to non-specifically interact with the selection surface. Of the 64 EPGPE clones screened from four rounds of display and selection, many were shown to have poor or non-specific binding, but one scFv was characterised that was affinity matured 2.6-fold over anti-GPE wild type affinity for IGF-I.A DNA shuffling method was developed to produce libraries of chimaeric scFvs between anti-GPEand NC10 (anti-neuraminidase scFv) with the objective of isolating functional IGF-I-bindingchimaeras. The NC10 scFv had its CDRs replaced with the anti-GPE CDRs prior to the shuffling toincrease the likelihood of isolating IGF-I binders. Ribosome display was used for selection from the chimaera libraries. Selection strategies included elution of specific binders by GPE peptide and a GPE 10-mer peptide. Selection was also performed using IGF-I immobilised on a BIAcore sensorchip as aselection surface. Again, much non-specific selection was observed as seen for display of EPGPE, for what was expected to be the same reasons. Selected scFvs were genuinely chimaeric but with poor expression and solubility and mostly non-specific in their binding. One characterised selected chimaera, made up of three segments of each of the parental scFvs, was shown to bind specifically to IGF-I by BIAcore. Steps to improve the efficiency of the ribosome display system have been identifiedand are discussed.
机译:本文描述了结合胰岛素样生长因子(IGF-1)的单链Fv(scFv)的构建和表征,以及该scFv作为模型蛋白在DNA改组和核糖体展示技术研究中的应用。抗体工程。从产生单克隆抗体的杂交瘤细胞株中分离出可变域基因,并通过PCR成功地连接以构建抗FPE的scFv。该基因的测序显示出异常短的重链CDR2区域。克隆的scFv在大肠杆菌中表达并纯化。表达水平低,蛋白质的溶解性差,最可能是由于CDR2缩写引起的折叠效率降低。使用BIAcore 1000上的表面等离振子共振分析了蛋白质的纯化单体形式与IGF-I的结合,该抗体的IgG版本对IGF-I-Gly-Pro-Glu的三个N末端残基具有特异性-转载。 scFv的解离常数为3.68 µM,对抗体的亲和力很低,比抗体的Fabversion计算的亲和力弱约36倍,但是可以得出结论,对scFv的计算的亲和力显然是由于凝胶过滤纯化的单体峰中存在非功能性或折叠错误的scFv物种,对真实亲和力的估计不足。具有在CDR2中插入的残基以使其恢复正常长度的抗GPE突变体产生了一种蛋白质,该蛋白质具有改善的表达和溶解性,同时保留了IGF-I结合。结论是shortCDR2是由于体细胞突变过程中产生的缺失而引起的,并为此建立了模型。开发了一种以兔网状细胞裂解物为核糖体来源的核糖体展示方法,以特异性选择针对IGF-I的抗GPE。 。容易出错的PCR用于产生抗GPE的随机点突变文库(EPGPE)。这是通过几个展示和选择周期完成的,但是通常观察到选择非特异性结合的scFv。这可能是由于所用系统中核糖体展示的蛋白质折叠不佳,可能是由于裂解物中存在DTT和/或抗GPE框架在保持稳定性的同时耐受突变的能力低所致。假定错误折叠,暴露疏水区域,将具有与选择表面非特异性相互作用的趋势。从四轮展示和选择中筛选出的64个EPGPE克隆中,许多克隆显示出较弱的或非特异性的结合,但其中一个scFv的特征是其对IGF-IA DNA的抗GPE野生型亲和力成熟了2.6倍。开发了一种改组方法以产生抗GPE和NC10之间的嵌合scFv文库(抗神经氨酸酶scFv),目的是分离功能性IGF-I-结合嵌合体。在改组之前,NC10 scFv的CDR被抗GPE CDR取代,以增加分离IGF-1结合物的可能性。核糖体展示用于从嵌合体文库中选择。选择策略包括通过GPE肽和GPE 10-mer肽洗脱特定的结合剂。还使用固定在BIAcore传感器芯片上的IGF-I作为选择表面进行选择。再次,由于预期是相同的原因,观察到许多非特异性选择,如用于EPGPE的展示。所选的scFvs确实是嵌合的,但表达和溶解性差,并且在结合方面大多非特异性。由BIAcore显示,由每个亲本scFv的三个片段组成的一个特征化的选定嵌合体特异性结合IGF-1。已经确定并讨论了提高核糖体展示系统效率的步骤。

著录项

  • 作者

    Roberts Anthony Simon;

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  • 年度 2004
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  • 原文格式 PDF
  • 正文语种 {"code":"en","name":"English","id":9}
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