The nucleotides remaining in the codon after cutting the vector with the given enzyme are (S-not for 5'-juncture because of stop codon in frame): BamHI-2, ClaI-3S, EcoRI-2, HindIII-2S, SalI-2S, XbaI-2S. Restriction digests of the clone give the following sizes (kb): BamHI--3.8; EcoRI--3.8; HindIII--3.8. Fusion proteins are produced in Escherichia coli usually in an insoluble form which facilitates purification. Hybrid proteins larger than 90 kDa are not expressed as efficiently as shorter ones. To prevent recircularization of the vector, the depositors recommend including 30 - 50 U of calf alkaline phosphatase in the restriction enzyme digestion used to prepare the vector. Escherichia coli containing pATH vectors should be grown on medium supplemented with tryptophan to prevent expression. Storage as DNA at -20C is preferred; long term storage as cells may result in selection of non-expressing promoter mutations. One of a series of vectors (ATCC 37695-37703) designed to facilitate expression of an open reading frame as a trpE fusion protein. Nucleotide 1 is the first in the trp fragment, at the beginning of a PvuII site 261 nucleotides upstream of the transcription start site. The multiple cloning site follows codon 323 of trpE. pATH1 was constructed from the following: 1-1391, the trp promoter and most of trpE; 1392-1455, multiple cloning sequence; 1456-3779, ClaI/PvuII fragment of pBR322, including bla (1696-2556) and ori. The EcoRI site was altered. |
