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| 产品名称： | pManflag20 CYT0024P, SCRF 387.0 |
|---|---|
| 商品货号： | TS199862 |
| Designations： | pManflag20 CYT0024P, SCRF 387.0 |
| GenBank Number： | M81110 |
| Species： | Saccharomyces cerevisiae Meyen ex E.C. Hansen |
| Depositors： | C Wong |
| Applications： | produces protein alpha-1,2-mannosyltransferase |
| Vector： | Construct size (kb): 6.800000190734863 |
| Insert： | DNA: genomic Insert information: Insert size (kb): 1.4 Genexa0: alpha-1, 2-mannosyltransferase, MNT1 DNAxa0: genomic Sourcexa0: Saccharomyces cerevisiae Genbank accessionxa0: M81110 Insert endsxa0: 5x92 XbaI, 3x92 SalI Insert lengths(kb): 1.399999976158142 Gene product: alpha-1,2-mannosyltransferase MNT1 Target Gene: alpha-1,2-mannosyltransferase |
| Insert Size (kb)： | 1.400 |
| Media： | ATCC® Medium 1227: LB Medium (ATCC medium 1065) with 50 mcg/ml ampicillin |
| Biosafety Level： | 1
Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country. |
| Shipping Information： | Escherichia coli containing the phagemid. |
| Comments： | 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. |
| Classification： | Saccharomycetes, Saccharomycetidae, Saccharomycetales, Saccharomycetaceae, Saccharomycetaceae, Saccharomyces, cerevisiae |
| References： | Wang P, et al. Enzymes in oligosaccharide synthesis: active-domain overproduction, specificity study and synthetic use of an alpha-1,2-mannosyltransferase with regeneration of GDP-Man. J. Org. Chem. : submitted, 1993. |
| Disclosure： | This material is cited in a US or other Patent and may not be used to infringe the claims. Depending on the wishes of the Depositor, ATCC may be required to inform the Patent Depositor of the party to which the material was furnished. This material may not have been produced or characterized by ATCC. |