Arnold C E, Parekh R N, Yang W, Wittrup K D
Department of Chemical Engineering, University of Illinois, Urbana, Illinois 61801, USA.
Biotechnol Bioeng. 1998 Aug 5;59(3):286-93.
Secretion of bovine pancreatic trypsin inhibitor (BPTI) in Saccharomyces cerevisiae was examined with four different leader peptides: the invertase signal peptide, the mfalpha1 signal peptide, a synthetic signal peptide, and a synthetic pre pro leader. BPTI secretion from a low-copy CEN plasmid varies from 1.8 to 10.4 microgram/mL among these constructs. Secretion titers correlate with dependence on signal recognition particle (SRP), with greatest secretion from the most SRP-dependent construct. Examination of co- vs post-translational translocation pathways and overall translocation efficiency by ubiquitin translocation assay (UTA) does not provide insight into the variation in BPTI secretion efficiency, perhaps due to alteration in translocation kinetics from the additional polypeptide fusion required by the assay. BPTI translocation efficiency (as measured by UTA) is found to drop markedly upon depletion of Srp54p, prior to any observable growth defect. Subsequent to stress response induction and the onset of slow growth (15-h doubling time), BPTI translocation efficiency recovers to the level observed prior to SRP depletion.
利用四种不同的前导肽对酿酒酵母中牛胰蛋白酶抑制剂（BPTI）的分泌情况进行了检测，这四种前导肽分别是：转化酶信号肽、mfalpha1信号肽、一种合成信号肽以及一种合成前原导肽。在这些构建体中，来自低拷贝CEN质粒的BPTI分泌量在1.8至10.4微克/毫升之间变化。分泌滴度与对信号识别颗粒（SRP）的依赖性相关，SRP依赖性最强的构建体分泌量最大。通过泛素转运测定法（UTA）对共翻译与翻译后转运途径以及整体转运效率进行检测，并未揭示BPTI分泌效率变化的原因，这可能是由于该测定法所需的额外多肽融合导致转运动力学发生改变。在未出现任何明显生长缺陷之前，当Srp54p耗尽时，发现BPTI转运效率（通过UTA测量）显著下降。在应激反应诱导和生长缓慢（倍增时间为15小时）开始后，BPTI转运效率恢复到SRP耗尽前观察到的水平。
