The pURI family of expression vectors: a versatile set of ligation independent cloning plasmids for producing recombinant His-fusion proteins.
A family of restriction enzyme- and ligation-independent cloning vectors has been developed for producing recombinant His-tagged fusion proteins in Escherichia coli. These are based on pURI2 and pURI3 expression vectors which have been previously used for the successful production of recombinant proteins at the milligram scale. The newly designed vectors combines two different promoters (lpp(p)-5 and T7 RNA polymerase Ø10), two different endoprotease recognition sites for the His₆-tag removal (enterokinase and tobacco etch virus), different antibiotic selectable markers (ampicillin and erythromycin resistance), and different placements of the His₆-tag (N- and C-terminus). A single gene can be cloned and further expressed in the eight pURI vectors by using six nucleotide primers, avoiding the restriction enzyme and ligation steps. A unique NotI site was introduced to facilitate the selection of the recombinant plasmid. As a case study, the new vectors have been used to clone the gene coding for the phenolic acid decarboxylase from Lactobacillus plantarum. Interestingly, the obtained results revealed markedly different production levels of the target protein, emphasizing the relevance of the cloning strategy on soluble protein production yield. Efficient purification and tag removal steps showed that the affinity tag and the protease cleavage sites functioned properly. The novel family of pURI vectors designed for parallel cloning is a useful and versatile tool for the production and purification of a protein of interest.
Laboratorio de Biotecnología Bacteriana, Instituto de Ciencia y Tecnología de Alimentos y Nutrición, CSIC, 28006 Madrid, Spain.
José Antonio
JA
Curiel
Blanca
B
de Las Rivas
José Miguel
JM
Mancheño
Rosario
R
Muñoz
D001483:Base Sequence; D002262:Carboxy-Lyases / Q000096:biosynthesis / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D010450:Endopeptidases / Q000235:genetics; D004926:Escherichia coli; D005822:Genetic Vectors; D006639:Histidine / Q000235:genetics; D048191:Lactiplantibacillus plantarum / Q000201:enzymology; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*
C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D010450:Endopeptidases; C099488:TEV protease; D002262:Carboxy-Lyases; C417714:phenolic acid decarboxylase