Ruiu L, Roque A C A, Taipa M A, Lowe C R
Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Avenida Rovisco Pais, 1049-001 Lisboa, Portugal.
J Mol Recognit. 2006 Jul-Aug;19(4):372-8. doi: 10.1002/jmr.782.
The protein surface is the interface through which a protein molecule senses the external world. The composition of this interface, in charged, polar and/or hydrophobic residues is crucial for both the activity and stability of the protein. Protein immobilization on surfaces has been extensively explored as one of the most effective approaches for stabilization. The mechanism of stabilization, however, is still poorly understood, and usually the success of any method is more a matter of trial and error rather than the result of rational concepts. The importance of local unfolding processes in a number of biologically significant processes has been recognized and attracted increasing attention. Unfolding regions have been localized in different proteins including the recombinant cutinase from Fusarium solani pisi. The study of three structural surface regions associated with early cutinase unfolding events was the basis for the approach followed in this work. A 64-member solid-phase combinatorial library of ligands was synthesized on a triazine-substituted agarose matrix using a modified 'mix and split' procedure. The combinatorial library was assessed for binding to cutinase from Fusarium solani pisi in a biologically active form. Four lead ligands (3/5, 3/7, 4/5, 4/7) have been selected in which immobilized cutinase presented a relative activity of 30-60% as compared to the free enzyme.
蛋白质表面是蛋白质分子感知外部世界的界面。该界面由带电荷、极性和/或疏水残基组成，对蛋白质的活性和稳定性都至关重要。蛋白质在表面的固定化作为最有效的稳定化方法之一已得到广泛探索。然而，稳定化的机制仍知之甚少，通常任何方法的成功更多是反复试验的结果，而非合理概念的成果。局部解折叠过程在许多生物学重要过程中的重要性已得到认可并日益受到关注。已在包括来自豌豆镰孢菌的重组角质酶在内的不同蛋白质中定位了解折叠区域。对与角质酶早期解折叠事件相关的三个结构表面区域的研究是本工作所采用方法的基础。使用改良的“混合与拆分”程序，在三嗪取代的琼脂糖基质上合成了一个由64个成员组成的配体固相组合文库。评估该组合文库与豌豆镰孢菌角质酶的生物活性形式的结合情况。已选择了四种先导配体（3/5、3/7、4/5、4/7），其中固定化角质酶与游离酶相比具有30 - 60%的相对活性。
