Highly efficient eukaryotic gene expression vectors for peptide secretion.
Recently, we constructed a series of highly efficient universal eukaryotic gene expression vectors (Sheay et al., BioTechniques 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 downstream of 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 transport of 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.
Chu
T H
TH
University of Medicine and Dentistry of New Jersey, Piscataway, USA.
Martinez
I
I
Olson
P
P
Dornburg
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D018388:Codon, Terminator / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D018388:Codon, Terminator; D008565:Membrane Proteins; D010455:Peptides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins