Highly Efficient Eukaryotic Gene Expression Vectors for Peptide Secretion

摘要

Recently, we constructed a series of highly efficient universal eukaryotic gene expression vectors (Sheay et al, BioTech-niques 15:856-862, 1993). Such vectors contain a viral promoter and enhancer followed by the adenovirus tripartite leader sequence, a multiple cloning site for the insertion of the gene of interest and a polyadenylation sequence. To enable expression of peptides to be secreted into the tissue culture medium or to be incorporated into the cell membrane, several modifications have been introduced into such vectors: stop codons in all three reading frames were inserted at the end of the multiple cloning site and a DNA sequence coding for a signal peptide for transport through the endoplasmatic reticulum (ER) was introduced downstreamof the adenovirus tripartite leader sequence followed by two unique restriction enzyme recognition sites, A protocol is described that allows fast and easy cloning of peptide-coding regions, i,e, PCR products, for expression and secretion. The transportof a genetically engineered chimeric transmembrane protein connected to this ER leader sequence was as efficient as that of the original protein from which the ER sequence has been derived. These universal vectors can also be used for the easy construction of any chimeric transmembrane or secretion proteins