Description: A Gateway destination vector for the production of recombinant proteins as fusions to the C-terminus of Pyrococcus furiosus maltodextrin-binding protein. Origin: A derivative of pET11c (Novagen) Replicon: ColE1 (pBR322) Antibotic Resistance: Ampicillin Promotor: bacteriophage T7 Inducer: IPTG (1 mM) Notes: The single cysteine in P. furiosus MBP (residue 33) was changed to a serine by site-directed mutagenesis. At the same time, a silent nucleotide substitution was introduced nearby to create a HindIII restriction site. Induction at 30 °C usually produces a greater yield of fusion protein than induction at 37 °C. Because it carries a gene (ccdB ) that encodes a lethal DNA gyrase poison, this vector must be propogated in an E. coli gyrA mutant such as DB3 or DB5 (Invitrogen), which is immune to the action of CcdB. Literature Citations: Fox, J. D., Routzahn, K. M., Bucher, M. H., and Waugh, D. S. (2003). Maltodextrin-binding proteins from diverse bacteria and archaea are potent solubility enhancers. FEBS Lett. 537: 53-57. Evdokimov, A. G., Anderson, D. E., Routzahn, K. M., and Waugh, D. S. (2001). Structural basis for oligosaccharide recognition by Pyrococcus furiosus maltodextrin-binding protein. J. Mol. Biol. 305: 891-904. Nucleotide Sequence:
Amino Acid Sequence: *
MKIEEGKVVI WHAMQPNELE VFQSLAEEYM ALSPEVEIVF EQKPNLEDAL KAAIPTGQGP DLFIWAHDWI GKFAEAGLLE PIDEYVTEDL LNEFAPMAQD AMQYKGHYYA LPFAAETVAI IYNKEMVSEP PKTFDEMKAI MEKYYDPANE KYGIAWPINA YFISAIAQAF GGYYFDDKTE QPGLDKPETI EGFKFFFTEI WPYMAPTGDY NTQQSIFLEG RAPMMVNGPW SINDVKKAGI NFGVVPLPPI IKDGKEYWPR PYGGVKLIYF AAGIKNKDAA WKFAKWLTTS EESIKTLALE LGYIPVLTKV LDDPEIKNDP VIYGFGQAVQ HAYLMPKSPK MSAVWGGVDG AINEILQDPQ NADIEGILKK YQQEILNNMQ GITSLYKKAG...passenger protein.* Amino acid residues encoded by the attR1 site in the Gateway cloning cassette are underlined.
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