A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide.
We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET), expression of a short 6XHis tag at N-terminus (pET3-His) and a high copy number of plasmid (pRSET). The small size of the vector (2.8 kb) and the high copy number/cell (200-250 copies) facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies) and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture). In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site) as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site). Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins.
Ramos
C R R
CR
Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brazil.
Abreu
P A E
PA
Nascimento
A L T O
AL
Ho
P L
PL
D001483:Base Sequence; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D014764:Viral Proteins
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases