Simonen M, Jämsä E, Makarow M
Institute of Biotechnology, University of Helsinki, Finland.
J Biol Chem. 1994 May 13;269(19):13887-92.
We have studied the relationship between folding and secretion competence of hsp150-beta lactamase fusion proteins in Saccharomyces cerevisiae. hsp150 is a secretory protein of yeast, and beta-lactamase was chosen, since its folding can be monitored by assaying its enzymatic activity. The hsp150 pre-pro-protein consists of a signal peptide, subunit I, a repetitive region, and a unique C terminus. Fusion of beta-lactamase to the C terminus of hsp150 produced Cla-bla protein, which was secretion-competent but inactive. The Pst-bla protein, where beta-lactamase was fused to subunit I, was also inactive and mostly secreted, but part of it remained in the pre-Golgi compartment. When beta-lactamase was fused to the C-terminus of the repetitive region, the fusion protein (Kpn-bla) was translocated to the endoplasmic reticulum, acquired disulfide bonds, and adopted an enzymatically active conformation. Kpn-bla was secreted to the medium without decrease of specific activity or retention in the cell. Folding of Kpn-bla to an active and transport-competent form required co-translational disulfide formation, since treatment of cells with dithiothreitol resulted in endoplasmic reticulum-retained inactive Kpn-bla. When dithiothreitol was removed, Kpn-bla resumed transport competence but remained inactive. Reduction of prefolded Kpn-bla did not inhibit enzymatic activity or transport. The repetitive hsp150 carrier may have use in heterologous protein production by conferring secretion competence to foreign proteins in yeast.
我们研究了酿酒酵母中hsp150-β-内酰胺酶融合蛋白的折叠与分泌能力之间的关系。hsp150是酵母的一种分泌蛋白，之所以选择β-内酰胺酶，是因为可以通过检测其酶活性来监测其折叠情况。hsp150前原蛋白由信号肽、亚基I、一个重复区域和一个独特的C末端组成。将β-内酰胺酶与hsp150的C末端融合产生Cla-bla蛋白，该蛋白具有分泌能力但无活性。Pst-bla蛋白（β-内酰胺酶与亚基I融合）也无活性且大部分被分泌，但其中一部分保留在高尔基体前区室中。当β-内酰胺酶与重复区域的C末端融合时，融合蛋白（Kpn-bla）被转运到内质网，获得二硫键，并形成具有酶活性的构象。Kpn-bla被分泌到培养基中，其比活性没有降低，也没有保留在细胞中。Kpn-bla折叠成有活性且具备转运能力的形式需要共翻译二硫键形成，因为用二硫苏糖醇处理细胞会导致内质网保留无活性的Kpn-bla。当去除二硫苏糖醇时，Kpn-bla恢复转运能力但仍无活性。对预折叠的Kpn-bla进行还原不会抑制酶活性或转运。重复的hsp150载体可能通过赋予酵母中外源蛋白分泌能力而用于异源蛋白生产。
