The cystine knot of a squash-type protease inhibitor as a structural scaffold for Escherichia coli cell surface display of conformationally constrained peptides
The cystine knot of a squash-type protease inhibitor as a structural scaffold for Escherichia coli cell surface display of conformationally constrained peptides
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DOI：
10.1093/protein/12.9.797
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发表时间：
1999-09-01
期刊：
影响因子：
--
通讯作者：
Kolmar, H
中科院分区：
文献类型：
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作者：
Christmann, A;Walter, K;Kolmar, H
Ecballium Eleterium胰蛋白酶抑制剂II（EETI-II）是蛋白酶抑制剂壁球家族的成员，由28个氨基酸残基组成，是胰蛋白酶的有效抑制剂。它的紧凑结构由三链的反平行β-片定义，该β-β-由三个形成胱氨酸结的分子内二硫键键合在一起。为了探索EETI-II肽的潜力作为表现随机寡肽的结构支架，我们构建了两个EETI-II衍生物，其中六分配抑制剂循环被13个沉积的Sentai Epitope取代病毒L蛋白质和通过人骨gla蛋白的17个保留表位，EETI-II和衍生变体是通过融合到麦芽糖结合蛋白雄性的。通过将融合到周质空间中的分泌，可以在高产量中获得完全氧化并正确折叠的EETI-II，EETI-II和衍生的变体可以通过融合到截短的LPP'-ompa融合到eScherichia coli外膜上：成熟脂蛋白的前九个残基以及跨越46-66蛋白残基的β链的膜。基因表达受到强且严格调节的TETA启动子/操作员的控制。发现细胞活力被LPP'-pompa'-eeti-II融合蛋白的高水平表达大大降低，以恢复细胞活力，外膜中融合蛋白的净积累通过引入琥珀色的水平降低至可耐受的水平。 LPP序列的位置9处的密码子，并利用琥珀抑制菌株作为表达宿主。通过间接免疫荧光证实了嵌入EETI-II CyStine Knot capfold的表位序列的细胞表面暴露，表达表达含有表位的EETI-II变体的细胞被证明是用各自的单克隆抗体标记的。通过将磁细胞分类与荧光激活的细胞排序相结合，可以丰富显示特定表位序列的细胞，这些结果表明E，E，大肠杆菌细胞表面的构型约束肽的构造肽的构造肽的表面显示为EETI-II cystine knot knot knot knot swot capfold有可能成为一种有效的技术，可以从与受体分子高亲和力结合的组合文库中快速分离小肽分子。
The Ecballium elaterium trypsin inhibitor II (EETI-II), a member of the squash family of protease inhibitors, is composed of 28 amino acid residues and is a potent inhibitor of trypsin. Its compact structure is defined by a triple-stranded antiparallel beta-sheet, which is held together by three intramolecular disulfide bonds forming a cystine knot. In order to explore the potential of the EETI-II peptide to serve as a structural scaffold for the presentation of randomized oligopeptides, we constructed two EETI-II derivatives, where the six-residue inhibitor loop was replaced by a 13-residue epitope of Sendai virus L-protein and by a 17-residue epitope from human bone Gla-protein, EETI-II and derived variants were produced via fusion to maltose binding protein MalE. By secretion of the fusion into the periplasmic space, fully oxidized and correctly folded EETI-II was obtained in high yield, EETI-II and derived variants could be presented on the Escherichia coli outer membrane by fusion to truncated Lpp'-OmpA: which comprises the first nine residues of mature lipoprotein plus the membrane spanning beta-strand from residues 46-66 of OmpA protein. Gene expression was under control of the strong and tightly regulated tetA promoter/operator. Cell viability was found to be drastically reduced by high level expression of Lpp'-OmpA'-EETI-II fusion protein, To restore cell viability, net accumulation of fusion protein in the outer membrane was reduced to a tolerable level by introduction of an amber codon at position 9 of the lpp' sequence and utilizing an amber suppressor strain as expression host. Cells expressing EETI-II variants containing an epitope were shown to be surface labeled with the respective monoclonal antibody by indirect immunofluorescence corroborating the cell surface exposure of the epitope sequences embedded in the EETI-II cystine knot scaffold. Cells displaying a particular epitope sequence could be enriched 10(7)-fold by combining magnetic cell sorting with fluorescence-activated cell sorting, These results demonstrate that E,coli cell surface display of conformationally constrained peptides tethered to the EETI-II cystine knot scaffold has the potential to become an effective technique for the rapid isolation of small peptide molecules from combinatorial libraries that bind with high affinity to acceptor molecules.