Protein engineering for conferring a biological function on ovalbumin

卵清蛋白是丝氨酸超家族的一员，但它不具有任何蛋白酶抑制活性。我们用定点突变的方法来赋予卵白蛋白丝氨酸的功能。我们通过X射线结晶学研究发现，卵白蛋白突变体R339T具有在弹性酶切割P1-P1‘后插入蛇纹环的能力。这对于我们的目标的实现来说是一个重要的发现，但突变体仍然没有对一种蛋白水解酶的抑制活性。为了进一步的诱变方法，我们创造了一个不同的卵清蛋白突变体R339T/A352R，其中P1-P1‘位点可以对抗胰蛋白酶。利用该突变体，建立了可靠的环插入率的高效液相色谱分析方法。由于丝氨酸的结构和功能状况，增加环插入率应该会导致抑制活性的获得。因此，我们在晶体结构数据的基础上进行了进一步的诱变，以加快环的插入速度。突变体K290T/A352R/R339T和R104/A352R/R339T以及二硫键还原的A352R/R339T分别比对照A352R/R339T的环插入率增加1.5、3.7和6.7倍。突变和二硫键还原应使远端折叠A结构具有更灵活的性质。因此，我们得出结论，蛇纹环插入反应的过渡态呈开放的片状A构象。

Ovalbumin is a member of serpin superfamily, but it does not have any proteinase inhibitor activity. We have done site-directed mutagenesis approach to confer serpin function on ovalbumin. We found by X-ray crystallography that an ovalbumin mutant R339T has an ability to undergo a serpin loop insertion after the P1-P1' cleavage by elastase. This was an important finding for the achievement of our aim, but the mutant did still not have an inhibitory activity against a protease. For further mutagenesis approach, we created a different ovalbumin mutant R339T/A352R in which the P1-P1' site is accessible against trypsin. Utilizing the mutant, a reliable HPLC analysis for the determination of the loop insertion rate was established. Because of the structural and functional situations of serpin, increased loop insertion rate should lead to the acquisition of the inhibitory activity. We therefore did further mutagenesis to accelerate the loop insertion rate on the basis of the data of crystal structure. Alternative mutants, K290T/A352R/R339T and R104/A352R/R339t, and the disulfide-reduced form of A352R/R339T, respectively, displayed 1.5, 3.7, and 6.7- fold increase in the loop insertion rate as compared A352R/R339T control. The mutation and disulfide reduction should give a more flexible nature on the distal sheet A structure. We therefore concluded that the transition state of the serpin loop insertion reaction assumes an opened sheet A conformation.