The DES-Inducible/Secreted Kit provides the vector pMT/BiP/V5-His for inducible, secreted expression of recombinant proteins and a choice of Blasticidin (pCoBlast) or hygromycin (pCoHygro) selection. This vector offers the inducible metallothionein promoter that is induced upon addition of copper sulfate or cadmium chloride. The N-terminal signal sequence from
the insect BiP gene is provided to direct the recombinant fusion protein through the secretory pathway of S2 cells into the culture medium. The pMT/BiP/V5-His vector offers the following additional features:
• Small size (3.6 kb) to improve DNA yields and increase subcloning efficiency
• C-terminal V5 epitope tag for rapid detection with Invitrogens Anti-V5 Antibody
• C-terminal 6xHis tag for simple purification of recombinant fusion proteins using nickel-chelating resin
To facilitate cloning, a set of three vectors-A, B, and C-is provided. Each vector has the multiple cloning site in a different reading frame relative to the BiP signal sequence.
pMT/BiP/V5-His is a 3.6 kb expression vector designed for use with the Drosophila Inducible/Secreted Expression System (DESÆ; Catalog nos. K4120-01 and K5120-01) available from Invitrogen. Upon transfection, the vector allows transient, inducible, secreted expression of your protein of interest in Drosophila cells. When cotransfected with the selection vector,
pCoHygro or pCoBlast, included with the appropriate DESÆ Inducible/Secreted Kit, pMT/BiP/V5-His allows selection of stable cell lines exhibiting inducible, secreted expression of the protein of interest. The vector contains the following elements:
• The Drosophila metallothionein (MT) promoter for high-level, metal-inducible expression of the gene of interest in S2 cells (Angelichio et al., 1991; Bunch et al., 1988; Maroni et al., 1986; Olsen, 1992)
• Drosophila BiP secretion signal for secreted expression of the gene of interest (Kirkpatrick et al., 1995)
• Multiple cloning site to facilitate cloning the gene of interest
• C-terminal peptide containing the V5 epitope and polyhistidine (6xHis) tag for detection and purification of your protein of interest (if desired)
• Three reading frames to facilitate in-frame cloning with the C-terminal peptide
• Ampicillin resistance gene for selection of transformants in E. coli
The control plasmid, pMT/BiP/V5-His/GFP, is included for use as a positive control for transfection and expression.
TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGT
AAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTA
ACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAA
GGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGG
CCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGT
TTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCAGTGAATTTTAACGTTGCAGGACAGGATGTGGTG
CCCGATGTGACTAGCTCTTTGCTGCAGGCCGTCCTATCCTCTGGTTCCGATAAGAGACCCAGAACTCCGGCC
CCCCACCGCCCACCGCCACCCCCATACATATGTGGTACGCAAGTAAGAGTGCCTGCGCATGCCCCATGTGCC
CCACCAAGAGTTTTGCATCCCATACAAGTCCCCAAAGTGGAGAACCGAACCAATTCTTCGCGGGCAGAACAA
AAGCTTCTGCACACGTCTCCACTCGAATTTGGAGCCGGCCGGCGTGTGCAAAAGAGGTGAATCGAACGAAAG
ACCCGTGTGTAAAGCCGCGTTTCCAAAATGTATAAAACCGAGAGCATCTGGCCAATGTGCATCAGTTGTGGT
CAGCAGCAAAATCAAGTGAATCATCTCAGTGCAACTAAAGGGGGGATCCGATCTCAATATGAAGTTATGCAT
ATTACTGGCCGTCGTGGCCTTTGTTGGCCTCTCGCTCGGGAGATCTCCATGGCCCGGGGTACCTACTAGTCC
AGTGTGGTGGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGGTCACCCATTCGAAGGTAAGCCTA
TCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTACCGGTCATCATCACCATCACCATTGAGTTTAAACCC
GCTGATCAGCCTCGACTGTGCCTTCTAAGGCCTGAGCTCGCTGATCAGCCTCGATCGAGGATCCAGACATGA
TAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTT
GTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATT
TTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGG
CTGATTATGATCAGTCGACCTGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAA
ATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAAT
GAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGC
TGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCA
CTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTAT
CCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAA
AGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTC
AGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTC
CTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATA
GCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCG
TTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGC
CACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGT
GGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCG
GAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGC
AGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGT
GGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAA
ATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAA
TCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGA
TAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGG
CTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCG
CCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACG
TTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCC
AACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCG
TTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCA
TGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGC
GACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCA
TCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAAC
CCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAA
GGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAAT
ATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAAC
AAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACAT
TAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGT