pET44a-CrtY

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The gene product of the carotenoid biosynthesis genes was expressed by subcloning crtY genes into the expression vector, pET44-a(+), which allows expression of a recombinant protein with a C-terminal fusion His-tag.	The expression plasmids were individually constructed under the control of the T7 promoter, and resulted in a pET44a(+)-CrtY plasmid (Fig. 1). This plasmid was transformed into the E. coli BL21(DE3) codon plus strain. The overexpression of crtY was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.05 mM at a cell density of OD600 = 0.5. The recombinant protein was overexpressed by adding IPTG and incubating at 25o C.

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The gene product of the carotenoid biosynthesis genes was expressed by subcloning crtY genes into the expression vector, pET44-a(+), which allows expression of a recombinant protein with a C-terminal fusion His-tag.
Protocol tips
The expression plasmids were individually constructed under the control of the T7 promoter, and resulted in a pET44a(+)-CrtY plasmid (Fig. 1). This plasmid was transformed into the E. coli BL21(DE3) codon plus strain. The overexpression of crtY was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.05 mM at a cell density of OD600 = 0.5. The recombinant protein was overexpressed by adding IPTG and incubating at 25o C.

Publication protocol

"The gene product of the carotenoid biosynthesis genes
was expressed by subcloning crtY genes into the expression vector, pET44-a(+), which allows expression of a recombinant protein with a C-terminal fusion His-tag. The crtY gene was amplified by PCR using a pair of forward oligonucleotides primer, CrtY-1 (5'-CATATGACCATGACGTGCTG-3'), and the reverse primer CrtY-2 (5'-CTCGAGTCATGCGTTTTCCTTCAG-3'), with the plasmid pCR-XL-TOPO-crtfull as a template, which carries the full-length astaxanthin biosynthesis gene cluster [10]. The PCR product was ligated into the vector pGEM-T (Promega, Madison, WI, USA). The subcloned plasmid was digested with NdeI and XhoI restriction enzymes, and the excised fragment was then ligated into the expression plasmid pET44-a(+) vector. The expression plasmids were individually constructed under the control of the T7 promoter, and resulted in a pET44a(+)-CrtY plasmid (Fig. 1). This plasmid was transformed into the E. coli BL21(DE3) codon plus strain.
The overexpression of crtY was induced by adding
isopropyl-β-D-thiogalactopyranoside (IPTG) to a final
concentration of 0.05 mM at a cell density of OD600 = 0.5. The recombinant protein was overexpressed by adding IPTG and incubating at 25o C. Temperature dependence of the solubility of proteins as a result of inclusion body formation has been reported for several recombinant proteins. Cultivating this E. coli transformant with the crtY gene at 37o C and lower temperatures showed that 25oC was the optimum growth temperature for obtaining soluble
lycopene cyclase in an active form. The yield at 25o
C was 35.7 mg of soluble protein (from 100 ml of cells), with a lycopene cyclase activity of 103 ng β-carotene ml-1 supernatant h-1."

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