Restriction digests of the clone give the following sizes (kb): XbaI/SalI--5.4, 1.3; EcoRI--6.3, 0.3, 0.2; BglII--6.8; HindIII--6.3, 0.3, 0.2; BamHI--6.8. The insert contains the following restriction sites (approximate kb from the 5' end): EcoRI--0.13, 0.41; HindIII--0.28; NcoI--0.16, 0.23, 0.78; PvuI--0.21; XmnI--1.22. Purification of the protein (all steps at 4C): Centrifuge 5 l of induced E. coli cells at 10,000 g for 10 min. Decant supernatant. Resuspend pellet in 200 ml of [20% sucrose, 10mM Tris-HCl pH 7.6]. Add 4 ml of 0.5 M EDTA and incubate on ice for 30 min. Centrifuge 5 min. Decant supernatant. Resuspend pellet in 20 ml cold distilled water. Incubate on ice for 30 min. Centrifuge 5 min. Carefully remove and save the supernatant, containing the periplasmic fraction. Slowly add 1 ml of [2 M Tris-HCl, 100 mM CaCl2.]. Incubate on ice for 10 minutes. Centrifuge 5 min. Dialyze supernatant 8 hr at 0C in 100 mM Tris-HCl, pH 7.6. Induce protein production using 0.005 mM IPTG for 12 hours at 30C (for proper protein folding and secretion into the periplasm). The insert corresponds to the coding sequence for the catalytic domain of the protein (nt 547-1782 of the sequence record). It was constructed by amplification from yeast DNA using primers incorporating XbaI (5') and SalI (3') sites: upstream 5'-ATATTTCTAGAAGAACTCAGCAATATATT-3' and downstream 5'-GCGCGTCGACTTATTACTCACGGAATTTTTTCCA-3'. Cell growth and gene induction: Grow to mid-log phase in M9 plus 2% casamino acids containing 1 mM CaCl2 and 100 ug/ml ampicillin at 37C. The order of the major features of this phagemid is: pMB1 ori - f1 ori - lacI - tac promoter - ompA - flag - XbaI/insert/SalI - rrnB terminator - ampR. |
