Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Patent
1994-02-10
1999-03-23
Patterson, Jr., Charles L.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
530417, 530422, 530425, 435189, C07K 114, C07K 116, C07K 136, C12N 902
Patent
active
058861572
ABSTRACT:
The invention provides a nucleic acid encoding a human cytochrome P450 2E1 comprising a 5' terminal deletion of 63 nucleotides, thereby encoding methionine at the first codon position of the 5' terminus, the codon ATG in the second codon position of the 5' terminus, and silent adenine and thymine nucleotide substitutions in the 5' region. The invention also provides a nucleic acid encoding a human cytochrome P450 2C10 comprising a 5' terminal deletion of nucleotides 7 through 60, thereby encoding methionine at the first codon position and alanine at the second position of the 5' terminus, and silent adenine and thymine nucleotide substitutions in the 5' region. The present invention also provides a method of purifying a recombinant cytochrome P450 protein from a host cell culture comprising the steps of: a. fractionating the host cells to prepare their membranes; b. adding a non-ionic detergent in a concentration of between 0.8% to 2% (w/v) and in a detergent to protein ratio of between 4:1 to 10:1 to the membranes; c. adding an ionic detergent in a concentration of between 0.4% to 0.8% (w/v) and in a detergent to protein ratio of between 2:1 to 4:1 to the membranes; d. centrifuging the membrane detergent mixture to remove insoluble materials; and, e. purifying the protein in the following order: i) through a diethylaminoethyl-beaded column; ii) through a carboxymethyl-beaded column; and iii) through a hydroxylapatite column. The invention also provides a purified recombinant human cytochrome P450 1A1 which has retained its catalytic activity, such as catalyzing 7-ethoxyresorufin O-deethylation and benzo(a)pyrene 3-hydroxylation.
REFERENCES:
patent: 5240831 (1993-08-01), Barnes
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Gillam Elizabeth M. J.
Guengerich F. Peter
Guo Zuyu
Sandhu Punam
Moore William W.
Patterson Jr. Charles L.
Vanderbilt University
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