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AssigneeSchering Corporation
InventorsGerhard Siewert, Werner Boidol, Joachim Daum
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PhoA mutant E. coli SB44, prepared by mutation of E. coli HB101 with an N-nitroso compound, can be used to identify and isolate recombinant plasmids into which a phoA gene has been incorporated. These plasmids can be used to transform bacteria which can be cloned and incubated to provide alkaline phosphatase in high yield. Moreover, these plasmid vectors can be modified in various ways so that the N-terminal amino acid sequence of phoA is followed in reading phase by the DNA coding for some other protein. In turn, these new plasmids can be used to transform bacteria which can be cloned and incubated to produce fusion proteins comprising the desired other protein in high yield and outside of the cell membrane in the periplasmatic space.
Substantially biologically pure phoA negative type mutant E. coli SB44 (DSM 1606), which is substantially incapable of in vivo recombination.
A process for preparing substantially biologically pure phoA negative type mutant E. coli SB44 (DSM 1606) having a mutation in the structural gene (phoA) for alkaline phosphatase and which is substantially incapable of in vivo recombination, comprising contacting substantially biologically pure E. coli K12 HB101 (DSM 1606) with an N-nitroso compound and subsequently separating the substantially biologically pure phoA mutant E. coli SB44 from any concomitantly-produced phoB mutants mutated in a regulatory gene (phoB).
A method of isolating plasmids containing phoA genes from a mixture of plasmids, some without and one or more with a phoA gene, comprising transforming substantially biologically pure phoA negative type mutant E. coli SB44 (DSM 1606), which is substantially incapable of in vivo recombination, with the mixture of plasmids, identifying phosphatase-positive colonies from the transformation mixture and isolating plasmid-DNA containing a phoA gene from these colonies.
A method for preparing the plasmid pSB86, comprising reacting to completion the plasmid pSB53 with PstI, isolating the thus-produced DNA fragment of about 2157 base pairs containing the N-terminal end of the phoA gene, partially cleaving the fragment with EcoRI, and coupling the resultant subfragment containing the N-terminal of the phoA gene with the vector plasmid pBR322.
A method for preparing the plasmid PSB53, comprising cutting the antibiotic-resistant vector plasmid pSB18 and the pho A plasmid pSB47 or pSB50 with the restriction endonucleases BamHI and Hind III to form a mixture of DNA;
A method for preparing plasmids containing a phoA gene, comprising reacting an antibiotic-resistant plasmid and chromosomal DNA from a wild-type bacteria with a restriction endonuclease, linking the terminal sites with DNA ligase to form a recombinant DNA, transforming substantially biologically pure phoA mutant E. coli SB44 (DSM 1606) with the DNA mixture, identifying phosphatase-positive colonies from the transformation mixture and isolating plasmid-DNA containing a phoA gene from these colonies.
A substantially biologically pure plasmid vector containing a first DNA sequence which continuously sequentially codes only a portion of the polypeptide sequence of an alkaline phosphatase, said polypeptide sequence containing the N-terminal amino acid of the alkaline phosphatase and the length of said portion being sufficient that the fusion protein coded by the combination of said DNA sequence and the adjacent sequence in reading phase therewith, will be transported into the periplasmatic space upon culturing of a bacterial cell transformed with said plasmid vector under effective culturing conditions.
A bacterium transformed with a plasmid vector containing a first DNA sequence which continuously sequentially codes only a portion of the polypeptide sequence of an alkaline phosphatase, said polypeptide sequence containing the N-terminal amino acid of the alkaline phosphatase and the length of said portion being sufficient that the fusion protein coded by the combination of said DNA sequence and the adjacent sequence in reading phase therewith will be transported into the periplasmatic space upon culturing of a bacterial cell transformed with said plasmid vector under effective culturing conditions.
A bacterial culture comprising a cloned bacterium transformed with a plasmid vector containing a first DNA sequence which continuously sequentially codes only a portion of the polypeptide sequence of an alkaline phosphatase, said polypeptide sequence containing the N-terminal amino acid of the alkaline phosphatase and the length of said portion being sufficient that the fusion protein coded by the combination of said DNA sequence and the adjacent sequence in reading phase therewith will be transported into the periplasmatic space upon culturing of a bacterial cell transformed with said plasmid vector under effective culturing conditions.
E. coli Sb44 transformed with plasmid pSB47.
E. coli SB44 transformed with plasmid pSB50.
E. coli SB44 transformed with plasmid pSB51.
E. coli SB44 transformed with plasmid pSB53.
E. coli HB101 transformed with plasmid pSB47.
E. coli HB101 transformed with plasmid pSB50.
E. coli K12 transformed with plasmid pSB47.
E. coli K12 transformed with plasmid pSB50.
E. coli transformed with plasmid pSB51.
S. marcescens wild-type transformed with plasmid pSB51.
S. marcescens wild-type transformed with plasmid pSB53.
E. coli SB44 transformed with plasmid PSB60.
E. coli SB44 transformed with plasmid pSB90.
Dependencies are inferred from the claim text.