Serpin B1 regulates the innate immune system by inhibiting serine and cysteine proteases that control programmed cell death and proliferation pathways. To provide recombinant human proteins for in vitro and in vivo studies we expressed and purified wild-type human serpin B1 and a C344A variant in the yeast S. cerevisiae. Both proteins expressed well and inhibited elastase and chymotrypsin. However, purification of wild-type serpin B1 in the absence of a reducing agent resulted in the specific loss of elastase - but not chymotrypsin - inhibition, concomitant with the formation of two higher molecular weight forms of the protein - a modified monomer and a dimer created via an intermolecular disulfide bond formed between C344 in respective serpin B1 monomers. In contrast to fully reduced serpin B1, both modified forms were good elastase substrates and catalytically cleaved at multiple adjacent sites within the reactive site loop. In contrast, purification of the C344A variant in the absence of a reducing agent yielded only one form of the protein which retained elastase and chymotrypsin inhibitory properties when purified. Furthermore, the elastase inhibitory activity of wild-type serpin B1, but not the C344A variant, was sensitive to oxidation. Thus, wild-type human serpin B1 should be formulated with a pharmaceutically acceptable reducing agent to protect C344 against post-translational oxidative modifications. Alternatively, the C344A variant of this protein may prove to be a suitable drug development candidate. These findings also suggest that inactivation of serpin B1 by oxidation may have a physiological role to play during inflammation.

中文翻译：

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重组人丝氨酸蛋白酶抑制剂B1的表达和纯化产生具有改变的蛋白酶抑制活性的新型分子：功能含义。

丝氨酸蛋白酶抑制剂B1通过抑制控制程序性细胞死亡和增殖途径的丝氨酸和半胱氨酸蛋白酶来调节先天免疫系统。为了提供用于体外和体内研究的重组人蛋白质，我们在酿酒酵母中表达和纯化了野生型人丝氨酸蛋白酶抑制剂B1和C344A变体。两种蛋白均表达良好，并抑制弹性蛋白酶和胰凝乳蛋白酶。但是，在没有还原剂的情况下纯化野生型丝氨酸蛋白酶抑制剂B1会导致弹性蛋白酶的特异性损失，但不能引起胰凝乳蛋白酶，但会导致蛋白质的两种更高分子量形式的形成，即修饰的单体和二聚体通过在相应的丝氨酸蛋白酶抑制剂B1单体中的C344之间形成的分子间二硫键产生的芳基。与完全减少的Serpin B1相比，两种修饰形式都是好的弹性蛋白酶底物，并且在反应位点环的多个相邻位点被催化裂解。相反，在不存在还原剂的情况下纯化C344A变体仅产生一种形式的蛋白质，该蛋白质在纯化时保留了弹性蛋白酶和胰凝乳蛋白酶的抑制特性。此外，野生型丝氨酸蛋白酶抑制剂B1（而不是C344A变体）的弹性蛋白酶抑制活性对氧化敏感。因此，野生型人丝氨酸蛋白酶抑制剂B1应与药学上可接受的还原剂一起配制，以保护C344免受翻译后氧化修饰的影响。或者，该蛋白的C344A变体可以证明是合适的药物开发候选物。