Short M K, Jeffrey P D, Kwong R F, Margolies M N
Department of Surgery, Massachusetts General Hospital, Boston 02114, USA.
J Biol Chem. 1995 Dec 1;270(48):28541-50. doi: 10.1074/jbc.270.48.28541.
We constructed a bacteriophage-displayed library containing randomized mutations at H chain residues 30-35 of the anti-digoxin antibody 26-10 Fab to investigate sequence constraints necessary for high affinity binding in an antibody of known crystal structure. Phage were selected by panning against digoxin and three C-16-substituted analogues. All antigen-positive mutants selected using other analogues also bound digoxin. Among 73 antigen-positive clones, 26 different nucleotide sequences were found. The majority of Fabs had high affinity for digoxin (Ka 3.4 x 10(9) M-1) despite wide sequence diversity. Two mutants displayed affinities 2- and 4-fold higher than the parental antibody. Analysis of the statistical distribution of sequences showed that highest affinity binding occurred with a restricted set of amino acid substitutions at positions H33-35. All clones save two retained the parental Asn-H35, which contacts hapten and hydrogen bonds to other binding site residues in the parental structure. Positions H30-32 display remarkable diversity, with 10-14 different substitutions for each residue, consistent with high affinity binding. Thus complementarity can be retained and even improved despite diversity in the conformation of the N-terminal portion of the H-CDR1 loop.
我们构建了一个噬菌体展示文库，该文库在抗地高辛抗体26 - 10 Fab的重链残基30 - 35处含有随机突变，以研究已知晶体结构抗体中高亲和力结合所需的序列限制。通过用地高辛和三种C - 16取代类似物进行淘选来筛选噬菌体。使用其他类似物筛选出的所有抗原阳性突变体也与地高辛结合。在73个抗原阳性克隆中，发现了26种不同的核苷酸序列。尽管序列多样性很大，但大多数Fab对 地高辛具有高亲和力（Ka 3.4 x 10(9) M-1）。两个突变体的亲和力比亲本抗体高2倍和4倍。对序列的统计分布分析表明，在H33 - 35位置发生一组受限的氨基酸取代时，会出现最高亲和力结合。除了两个克隆外，所有克隆都保留了亲本的Asn - H35，该残基在亲本结构中与半抗原接触并与其他结合位点残基形成氢键。H30 - 32位置表现出显著的多样性，每个残基有10 - 14种不同的取代，这与高亲和力结合一致。因此，尽管H - CDR1环N端部分的构象存在多样性，但互补性仍可保留甚至得到改善。
