McConnell S J, Uveges A J, Fowlkes D M, Spinella D G
Cytogen Corporation, Princeton, NJ 08540-5237, USA.
Mol Divers. 1996 May;1(3):165-76. doi: 10.1007/BF01544954.
We have constructed two phage display libraries expressing N-terminal pIII fusions in M13 composed of 37 and 43 random amino acid domains, respectively. The D38 library expresses 37 random amino acids with a central alanine residue, and the DC43 library contains 43 random amino acids with a central cysteine flanked by two glycine residues, giving the displayed peptide the potential to form disulfide loops of various sizes. We demonstrate that the majority of random sequences in both libraries are compatible in pentavalent display with phage viability. The M13 phage display vector itself has been engineered to contain a factor Xa protease cleavage site to provide an alternative to acid elution during affinity selection. An in-frame amber mutation has been inserted between the pIII cloning sites to allow for efficient selection against nonrecombinant phage in the library. These libraries have been panned against mAb 7E11-C5, which recognizes the prostate-specific membrane antigen (PSM). Isolated phage display a consensus sequence that is homologous to a region in the PSM molecule.
我们构建了两个噬菌体展示文库，分别在M13中表达由37个和43个随机氨基酸结构域组成的N端pIII融合蛋白。D38文库表达37个随机氨基酸，中间有一个丙氨酸残基；DC43文库包含43个随机氨基酸，中间有一个半胱氨酸，两侧各有一个甘氨酸残基，这使得展示的肽有可能形成各种大小的二硫键环。我们证明，两个文库中的大多数随机序列在五价展示中与噬菌体活力兼容。M13噬菌体展示载体本身经过改造，含有一个因子Xa蛋白酶切割位点，以便在亲和选择过程中提供一种替代酸洗脱的方法。在pIII克隆位点之间插入了一个框内琥珀突变，以有效筛选文库中的非重组噬菌体。这些文库已针对识别前列腺特异性膜抗原（PSM）的单克隆抗体7E11-C5进行淘选。分离出的噬菌体展示出与PSM分子中一个区域同源的共有序列。
