Jose Joachim, Zangen Dirk
Bioanalytics, Institute for Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf, Germany.
Biochem Biophys Res Commun. 2005 Aug 12;333(4):1218-26. doi: 10.1016/j.bbrc.2005.06.028.
The Kunitz type protease inhibitor aprotinin, containing three intramolecular disulfide bonds, was expressed on the surface of Escherichia coli by Autodisplay. For this purpose, the aprotinin gene was fused in-frame to the transporter domain encoding DNA region of the AIDA-I autotransporter protein. Culture of cells supplied with the artificial gene at reducing conditions resulted in the translocation of aprotinin to the cell surface. Correct folding of aprotinin was shown by high affinity to its target enzyme HLE. No surface translocation was detectable under non-reducing conditions, indicating the degradation of aprotinin in the periplasm. By the use of periplasmic-protease defective E. coli strains PW147, PW151, and PW152, under non-reducing conditions, significant amounts of aprotinin appeared in the periplasm but not at the surface. Our results indicate that aprotinin molecules, reaching stable conformation before transport across the outer membrane, are degraded in the periplasm due to proteolysis. In case folding can be prevented, i.e., by blocking disulfide bond formation in the periplasm, aprotinin is translocated and can adopt its active conformation at the cell surface.
含有三个分子内二硫键的库尼茨型蛋白酶抑制剂抑肽酶，通过自展示技术在大肠杆菌表面表达。为此，将抑肽酶基因与AIDA-I自转运蛋白的转运结构域编码DNA区域读码框融合。在还原条件下培养携带人工基因的细胞，导致抑肽酶转运至细胞表面。抑肽酶对其靶酶HLE的高亲和力表明其折叠正确。在非还原条件下未检测到表面转运，表明抑肽酶在周质中降解。通过使用周质蛋白酶缺陷型大肠杆菌菌株PW147、PW151和PW152，在非还原条件下，周质中出现了大量抑肽酶，但细胞表面没有。我们的结果表明，抑肽酶分子在穿过外膜之前达到稳定构象，但由于蛋白水解作用在周质中被降解。如果能够防止折叠，即通过阻断周质中二硫键的形成，抑肽酶就会转运并在细胞表面形成其活性构象。
