Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
2007-08-28
2007-08-28
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C435S252300, C435S254110, C435S254220, C435S320100, C536S023200
Reexamination Certificate
active
10498697
ABSTRACT:
The invention provides modification to the polynucleotide coding sequence forPichia angustaNAD (P)H: nitrate reductase [YNaR1; GenBank accession number Z49110], which has Enzyme Commission number 1.7.1.2 (formerly EC 1.6.6.2), yielding the polynucleotide coding sequence for simplified eukaryotic nitrate reductase (S-NaR1). The invention also provides a method for recombinant expression of said polynucleotide code in the cells of the methylotrophic yeastPichia pastoristo produce the polypeptide for S-NaR1, which binds the host-produced molybdenum-molybdopterin cofactor and intracellularly forms catalytically active, nitrate-reducing enzyme as small and stable multimeric proteins. The invention also provides a method for rapid and high-yielding purification of S-NaR1 by utilizing the hexa-histidine sequence at the carboxyl-terminus of said polypeptide for immobilized metal affinity chromatography.
REFERENCES:
patent: 5612198 (1997-03-01), Brierley et al.
International Search Report, PCT/US02/39341, Jul. 11, 2003, 4 pgs, ISA/US.
Cloning and Disruption of the YNR1 Gene Encoding the Nitrate Reductase Apoenzyme of the Yeast Hansenula, Avila et al., FEBS Letters 366, 1995, pp. 137-142.
Nitrate and Nitrate Control of Respiratory Nitrate Reduction in Denitrifying Pseudomonas Stutzeri by a Two-Component Regulatory System Homologous to NarXL ofEscherichia coli, Härtig et al., Journal of Bacteriology, vol. 181, No. 12, Jun. 1999, pp. 3658-2665.
Barbier Guillaume G
Campbell Ellen R
Campbell Wilbur H
Fish & Associates PC
Nitrate Elimination Co., Inc.
Slobodyansky Elizabeth
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