Blondel A, Bedouelle H
Unité de Biochimie Cellulaire (Unité de Recherche associée D1129 du Centre National de la Recherche Scientifique), Institut Pasteur, Paris, France.
Eur J Biochem. 1990 Oct 24;193(2):325-30. doi: 10.1111/j.1432-1033.1990.tb19341.x.
A hybrid between the maltose-binding protein (MalE) of Escherichia coli and the gene 5 protein (G5P) of phage M13 was constructed at the genetic level. MalE is a monomeric and periplasmic protein while G5P is dimeric and cytoplasmic. The hybrid (MalE-G5P) was synthesized in large amounts from a multicopy plasmid and efficiently exported into the periplasmic space of E. coli. The export was dependent on the integrity of the signal peptide. MalE-G5P was purified from a periplasmic extract by affinity chromatography on cross-linked amylose, with a yield larger than 50,000 molecules/E. coli cell. The hybrid specifically bound denatured but not double-stranded DNA cellulose, as native G5P. Sedimentation velocity and gel-filtration experiments showed that MalE-G5P exists as a dimer. Thus, it was possible to efficiently translocate through the membrane a normally cytoplasmic and dimeric protein, by fusion to MalE. Moreover, the passenger protein kept its activity, specificity and quaternary structure in the purified hybrid. MalE-G5P will enable the study of mutant G5P that no longer binds single-stranded DNA and therefore cannot be purified by DNA-cellulose chromatography.
在基因水平上构建了大肠杆菌麦芽糖结合蛋白（MalE）与噬菌体M13基因5蛋白（G5P）的杂交体。MalE是一种单体周质蛋白，而G5P是二聚体细胞质蛋白。杂交体（MalE-G5P）从多拷贝质粒大量合成，并有效地输出到大肠杆菌的周质空间。这种输出依赖于信号肽的完整性。通过交联直链淀粉亲和层析从周质提取物中纯化MalE-G5P，产量大于50,000个分子/大肠杆菌细胞。该杂交体像天然G5P一样特异性结合变性但非双链的DNA纤维素。沉降速度和凝胶过滤实验表明MalE-G5P以二聚体形式存在。因此，通过与MalE融合，可以有效地将一种通常位于细胞质的二聚体蛋白转运穿过膜。此外，在纯化的杂交体中，客蛋白保留了其活性、特异性和四级结构。MalE-G5P将有助于研究不再结合单链DNA、因此不能通过DNA纤维素层析纯化的突变型G5P。
