A novel GFP-fused eukaryotic membrane protein expression system in lactococcus lactis and its application to overexpression of an Elongase.
Eukaryotic membrane proteins are often difficult to produce in large quantities, which is a significant obstacle for further structural and biochemical investigation. Lactococcus lactis has several properties that are ideal for enhanced expression of eukaryotic membrane proteins. In this article, a novel lactococcal vector was constructed, by inserting a green fluorescent protein (GFP) sequence into the vector pNZ8148. The resulting vector pKj-gfp was applied to the overexpression of an elongase-GFP fusion in L. lactis. After adding nisin, the expression of the fusion was confirmed via microscopy, the conditions were optimized by fluorescent spectroscopy, and the location of the fusion was monitored through in-gel fluorescence, indicating that the fusion was directed into the cytoplasmic membrane and no degradation was observed. The ELKJ-GFP fusion accounted for approximately 15% of the total membrane proteins using PDQuest software. These results indicate that the lactococcal system can be used for the overproduction and convenient detection of eukaryotic membrane proteins.
Niu
Yan
Y
State Key Laboratory for Microbial Technology, Shandong University, Shanda Nan Road 27, Jinan, 250100, China.
Kong
Jian
J
Xu
Yi
Y