Swimmer C, Lehar S M, McCafferty J, Chiswell D J, Blättler W A, Guild B C
ImmunoGen Inc., Cambridge, MA 02139.
Proc Natl Acad Sci U S A. 1992 May 1;89(9):3756-60. doi: 10.1073/pnas.89.9.3756.
We demonstrate that the B chain of ricin toxin preserves its lectin activity when expressed as a fusion protein on the surface of fd phage. Moreover, B chain, which folds into two topologically similar globular domains, can be dissected into amino-terminal and carboxyl-terminal domains to form single binding domains (SBDs) of B chain, each of which displays specificity for complex galactosides. The specific binding exhibited by the fusion protein of these SBDs was eliminated when amino acid substitutions Gly-46 in SBD1 or Gly-255 in SBD2 for native asparagine were introduced to alter key residues implicated in hydrogen bonding with substrate. These data demonstrate that it is possible to use a prokaryotic expression system to stably express and screen ricin B chain and its SBDs for sugar-binding mutants. Expression of ricin B chain on the surface of fd phage provides a method that can be used to efficiently select mutants with altered binding activities from a randomly generated library.
我们证明，蓖麻毒素的B链在fd噬菌体表面作为融合蛋白表达时，保留了其凝集素活性。此外，B链折叠成两个拓扑结构相似的球状结构域，可以被切割成氨基末端和羧基末端结构域，形成B链的单结合结构域（SBD），每个结构域对复合半乳糖苷都具有特异性。当将SBD1中的氨基酸残基Gly-46或SBD2中的Gly-255替换为天然天冬酰胺以改变与底物氢键形成相关的关键残基时，这些SBD融合蛋白所表现出的特异性结合被消除。这些数据表明，可以使用原核表达系统稳定表达和筛选蓖麻毒素B链及其SBD的糖结合突变体。蓖麻毒素B链在fd噬菌体表面的表达提供了一种方法，可用于从随机生成的文库中高效筛选具有改变的结合活性的突变体。
