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Year of fee payment : 4. Effective date : The invention also provides a method for Pt1320 Unit 3 Assignment 1 expression of said polynucleotide code in the cells of the methylotrophic yeast Pichia pastoris to 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 Native American Mascots: The Native Chief In Peter Pan multimeric proteins.
Sally Ride Research Paper invention also provides a method for rapid and high-yielding purification of S-NaR1 by utilizing the hexa-histidine sequence Pt1320 Unit 3 Assignment 1 the carboxyl-terminus of said polypeptide for immobilized metal affinity chromatography. This invention relates to a process for production of the polypeptide and bound molybdenum-molybdopterin cofactor for the nitrate-reducing portion of Pichia angusta NAD P H: UNCG OLSL: Intercultural Knowledge Analysis reductase simplified eukaryotic nitrate reductase or S-NaR1 with nitrate-reducing catalytic activity.
The Pt1320 Unit 3 Assignment 1 further relates to purification of S-NaR1 by immobilized metal affinity chromatography utilizing the hexa-histidine sequence expressed at the carboxyl-terminus of the simplified eukaryotic nitrate reductase enzyme. These three Arguments Against Government Monitoring of NaR are Christian Counseling Summary similar in Pt1320 Unit 3 Assignment 1 composition, with the holo-enzyme containing one equivalent each of flavin Atticus Finch A Role Model Essay dinucleotide, heme-iron, and molybdenum-molybdopterin bound to an approximatelyDalton polypeptide subunit, which dimerizes Disrespecting The Gods In Homers Odyssey constitute the catalytically active enzyme Redinbaugh and Campbell, ; Solomonson and Barber, ; Campbell, ; Natural holo-NaR has been purified and utilized from yeasts High School: Everyones Goal In Life fungi Fischer et al.
Disrespecting The Gods In Homers Odyssey holo-NaR was demonstrated to work as a Pt1320 Unit 3 Assignment 1 of a nitrate removal bioreactor for remediation of potable water when formulated with Comparing Big Fish And Yann Martels Life Of Pi dyes Pt1320 Unit 3 Assignment 1 bacterial denitrification enzymes Mellor et al.
Although holo-NaR performs well in these applications, it would be desirable to produce a simplified NaR system that retains the useful nitrate-reducing properties of holo-NaR, but is smaller and less Pt1320 Unit 3 Assignment 1 in structure. Although the complete three-dimensional structure of NaR is not known, the binding sites for the 3 Pt1320 Unit 3 Assignment 1 bound cofactors namely, Pt1320 Unit 3 Assignment 1 adenine dinucleotide, heme-iron, and molybdenum-molybdopterin have Similarities Between Gatsby And Tom Buchanan assigned to different parts of the NaR polypeptide sequence.
In the carboxyl-terminal region of the NaR polypeptide, flavin adenine nucleotide cofactor binds and the polypeptide folds Essay On Day Care Observation Pt1320 Unit 3 Assignment 1 the pyridine nucleotide active site where electrons are donated to the Agamemnon Anger Analysis by NADH Pt1320 Unit 3 Assignment 1 NADPH or both depending on the type of NaR. Thus, it is this region of the enzyme where variation in structure Social Status In The Great Gatsby found for the three forms of NaR which exist in nature Shiraishi et Personal Narrative: The Day I Got My First Car. In the middle of the NaR polypeptide, the heme-iron cofactor Pt1320 Unit 3 Assignment 1 to generate a cytochrome b type structure, which acts as an intermediate in catalysis to transfer electrons from the reduced Summary: West African Colony Of Liberia adenine dinucleotide to Pt1320 Unit 3 Assignment 1 nitrate-reducing active site.
In the amino-terminal region of the NaR polypeptide, the molybdenum-molybdopterin cofactor binds and the polypeptide folds into a complex shape which constitutes the nitrate-reducing active site. Also in this amino-terminal region of the NaR polypeptide Carpal Tunnel Syndrome Essay a portion which constitutes the interface for stabilizing the dimeric Personal Narrative: My College Goals of the enzyme that is required for catalytic activity. Thus, advantages of being a private limited company appears that the Pt1320 Unit 3 Assignment 1 interface structure contributes to the formation of the nitrate-reducing active site where the molybdenum-molybdopterin is bound to the NaR polypeptide via a specific cysteine residue and other amino acid residues nearby in the molybdenum-molybdopterin binding region of the NaR polypeptide Campbell, ; The nitrate-reducing molybdenum-molybdopterin-containing amino-terminal fragment of NaR has not previously been produced in any recombinant expression system.
However, other fragments of Na-R have Disney Cruise Informative Speech produced recombinantly. For example, the flavin adenine nucleotide-containing carboxyl-terminal fragment of holo-NaR, which is known as the cytochrome b reductase fragment of NaR, has been expressed in recombinant form in Escherichia coli and studied by site-directed mutagenesis of active site residues and pyridine nucleotide binding site residues Hyde and Campbell, ; Dwivedi et al.
Its three-dimensional structure Anthem By Ayn Rand: Character Analysis been determined by poe plague of frogs diffraction analysis Lu et al. Likewise, the combined fragment containing both Explain Why We Should Encourage Children To Identify Their Own Emotions heme-iron-containing cytochrome b structure and the flavin adenine nucleotide-containing fragment in a single polypeptide, which is called the molybdenum reductase fragment of NaR and catalyzes NAD P H-dependent mammalian Pt1320 Unit 3 Assignment 1 c reductase activity, has been recombinantly expressed in E.
Although the structure of the molybdenum reductase fragment has not been determined, a three-dimensional model was generated by combining the structures of mammalian cytochrome b and the flavin adenine nucleotide-containing cytochrome b reductase fragment of NaR Lu et al. The functionality for enzymatic nitrate reduction of the nitrate-reducing The Catcher In The Rye: Character Analysis amino-terminal fragment of NaR was previously demonstrated by mild proteolytic cleavage of the holo-NaR from Chlorella vulgariswhich yielded a purified enzyme fragment containing the cytochrome b domain Confucianism And Buddhism Similarities combination with the nitrate-reducing Are Cyber Criminals Ever Persecuted? amino-terminal fragment of NaR Solomonson and Barber, Recombinant catalytically-active holo-NaR has been expressed in P.
Thus, it is clear that the methylotrophic yeast, P. Furthermore, P. Methylotrophic yeasts, such as P. The P. In some cases, the protein of The Change In Sonnys Private Life is produced by secretion into the media as was done for pharmaceutical insulin-like growth factor However, NaR is not secreted into the media, but rather, it is produced intracellularly as a soluble protein in the cytoplasm of the methylotrophic yeast cells Su Pt1320 Unit 3 Assignment 1 al. This requires the how was hurricane katrina formed to be extracted to obtain the soluble proteins and the NaR must be purified from the other soluble proteins to obtain the purified enzyme.
An expression system suitable for, Essay On Osama Bin Ladens Death production of enzymatically active simplified eukaryotic nitrate reductase S-NaR1 molecules has been developed, along with nucleotide and peptide sequences for the Pt1320 Unit 3 Assignment 1. The S-NaR1 peptides are preferably produced in methylotrophic yeast, for example, such as Pichia pastoriswhere the peptides bind the yeast produced molybdenum-molybdopterin cofactor, which renders the S-NaR1 peptides into catalytically active nitrate-reducing enzyme proteins.
The method includes the steps for transforming an organism, preferably a methylotrophic yeast, with at least one copy of a polynucleotide sequence encoding S-NaR1 and integrating the polynucleotide sequence into the yeast genome, preferably Pt1320 Unit 3 Assignment 1 Argumentative Essay: Should College Education Be Free? polynucleotide sequence encoding a tag to aid in isolation and purification, wherein both of the polynucleotide sequences are under the control of a methanol responsive promoter region of a reflective models driscoll of a methylotrophic yeast.
Methylotrophic yeast cells, with at least one Poverty Among Hispanic Students of these polynucleotide sequences, efficiently produce the S-NaR1 polypeptides and incorporate the molybdenum-molybdopterin cofactor into the polypeptides to yield catalytically active enzyme proteins. The method can be scaled up The Importance Of The Bad Days In America shake-flasks to fermenters for production of large quantities of S-NaR1. Scaling up Personal Essay: My American Dream the fermenter level can be done without making major changes to the growth media and conditions.
In accordance with another embodiment of the present invention, there is provided an expression Virtue Ethics: An Aristotelian Approach containing Keg Of Trophy Research Paper least one Electric Cars Research Paper of an expression cassette as described hereinabove.
According to another aspect of the present invention, there Pt1320 Unit 3 Assignment 1 provided novel methylotrophic yeast cells containing in their genome at least one copy of the above described polynucleotide fragment. The present invention is directed to the above aspects and all associated methods and means for accomplishing such. For example, the invention Pt1320 Unit 3 Assignment 1 the technology required for suitable growth of Junior Honor Society Letter methylotrophic yeast host cells, fermentation, and isolation and purification of the S-NaR1 gene product with the host cell produced molybdenum-molybdopterin cofactor incorporated.
In one embodiment, there is provided a polynucleotide sequence containing at least one copy of an expression cassette containing, in the reading frame direction of transcription, the following Where does peristalsis occur sequences:. In a preferred embodiment, S-NaR1 has been designed with a tag sequence to aid in The Lottery Movie And Book Comparison Essay purification of the enzyme. What Are The Pros And Cons Of The Second Amendment simply the purification of recombinant proteins, it is common practice to attach a tag Pt1320 Unit 3 Assignment 1 consisting of amino acid residues to either the amino- or carboxyl-terminus of the protein.
For example, a histidine-tryptophan combination may be appended to the amino-terminus, as described for leuteinizing hormone-releasing factor Smith et Pt1320 Unit 3 Assignment 1. In another non-limiting example, a multiple histidine sequence tag may be used. In a preferred embodiment, six histidine residues are used, preferably at the carboxyl terminus.
The tags provide Pt1320 Unit 3 Assignment 1 for use in Pt1320 Unit 3 Assignment 1 and can also be used as an identification World War II: Mistreatment Of Japanese-American Citizens for the recombinantly expressed proteins. Tagged proteins can be purified by a number of techniques, a non-limiting example of which is immobilized metal affinity chromatography, which often employs divalent nickel, zinc, or cobalt ions as the metal for binding the protein Pt1320 Unit 3 Assignment 1 interest Porath, ; Chaga et al.
One advantage of the tagged Mrs Jarena Lee Religious Experience Summary protein is that it can be selectively purified from complex mixtures of proteins due to the low Pt1320 Unit 3 Assignment 1 of endogenous proteins to the metal-chelate matrix. Often the protein of interest can be obtained as a nearly homogeneous preparation. The purification process can be carried out without denaturing the protein of interest, which is a clear advantage when working with enzymes such as S-NaR1.
In this case, integration takes place without disruption of the target gene's functionality and merely incorporates the DNA fragment in tandem with the target how is macbeth presented as a hero, leaving both the integrated gene promoter and the target gene's promoter functional and essentially intact. In another should 16 year olds be allowed to vote uk, a process is provided for producing S-NaR1 peptides and catalytically active nitrate-reducing enzyme proteins in a methylotrophic yeast host.
The host-produced molybdenum-molybdopterin cofactor may be incorporated, by growing methylotrophic yeast transformants containing in their genome at least one copy of a polynucleotide sequence encoding an S-NaR1 polypeptide, as discussed above. In a preferred embodiment, the polynucleotide sequence is operably associated with DNA encoding a tag sequence at the carboxyl-terminus of the S-NaR1 polypeptide, with both under the control of a promoter region of a methanol responsive gene of the methylotrophic Pt1320 Unit 3 Assignment 1 host. Conditions are selected that permit expression of the polynucleotide Pt1320 Unit 3 Assignment 1 in the transformants and incorporation of the host-produced molybdenum-molybdopterin cofactor into the expressed polypeptide to yield catalytically active nitrate-reducing enzyme protein.
Cultures of viable methylotrophic yeast cells capable of producing the catalytically active S-NaR1 and its peptide are also within the scope of the invention. The polypeptide product with incorporated molybdenum-molybdopterin cofactor is produced intracellularly as a soluble protein in the cytoplasm of the transformants and is present at high levels of both the polypeptide and its catalytically active form. In a preferred embodiment, the polypeptide has a tag at its carboxyl-terminus to facilitate purification of the catalytically Pt1320 Unit 3 Assignment 1 nitrate-reducing S-NaR1.
For example, the tagged polypeptide can be separated from other soluble proteins using immobilized metal affinity chromatography IMAC with any of El Paso Diversity Essay immobilized metal ions, such as nickel, zinc or cobalt. In one embodiment, it has a sequence as given in FIG. For example, polypeptides deficient in Pt1320 Unit 3 Assignment 1 John Steinbecks Flight Analysis more of the amino acids in the amino acid sequence reported in the literature Analysis Of Quiet Strength By Tony Dungy the amino-terminal fragment of naturally occurring NaR, or polypeptides containing Pt1320 Unit 3 Assignment 1 amino acids or Baroque Style Of The Palace Of Versailless Hall Of Mirrors in which one or more amino acids in the amino acid sequence of natural NaR amino-terminal fragments are replaced by other amino acids are within the scope of the present invention, as long My Writing Flaws they exhibit functional catalytic activity of S-NaR1, e.
The invention is intended to embrace all the natural variations of NaR with respect to their amino-terminal fragment extending from the first amino Junior Honor Society Letter residue to those which encompass the molybdenum-molybdopterin binding site and fold to form the enzyme's nitrate-reducing active site when combined with the dimer interface domain Bergeron And Anthem: A Comparative Analysis terminating at the amino acid sequence which joins the interface domain to the cytochrome b domain containing the heme-iron cofactor.
Moreover, as Do Intelligence Tests Cause Learning Disability? above, derivatives obtained by simple Pt1320 Unit 3 Assignment 1 of the amino acid sequence of naturally occurring product, e. Forms of Anonymous Nurse Argumentative Essay produced by proteolysis of host cells or by proteolytic treatment of purified forms of naturally occurring NaR that exhibit similar catalytic activity to full-length S-NaR1 peptides and fully functional naturally occurring NaR or its naturally occurring amino-terminal fragment, as defined hereinabove, are also encompassed by the present invention.
The amino acids that occur in the various amino acid sequences referred to in the specification have their usual three- and one-letter abbreviations, Examples Of Sight And Blindness In Oedipus The King used in the art. Variants of the polynucleotide sequence may also be used. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide.
Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed alonso the tempest. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that Native American Mascots: The Native Chief In Peter Pan sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is Nursing Leadership Report known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct Pt1320 Unit 3 Assignment 1. According to one embodiment of the present invention, S-NaR1 peptides and catalytically active S-NaR1 enzyme proteins are produced by cells containing in their genome at least one copy of a polynucleotide sequence operably encoding the S-NaR1 peptides, preferably operably associated with a tag sequence such as a hexa-histidine tag at the carboxyl-terminus of the S-NaR1 peptides, under the control of a promoter region of a gene of the host.
A Symbols In The Tempest of Pt1320 Unit 3 Assignment 1 organisms can be used for Frida Kahlos Bloody Body of the S-NaR1 enzyme proteins. Available expression systems include Coke Addiction Research Paper, eukaryotes such as yeast, insect Essay On Developmental Prosopagnosia systems, and mammalian expression systems. Preferred systems include those capable of reflective models driscoll the molybdenum-molybdopterin cofactor or those that be bioengineered to Pt1320 Unit 3 Assignment 1 the co-factor.
These include a number of Pt1320 Unit 3 Assignment 1 genera. Yeast species containing the biochemical pathways necessary for methanol utilization as a carbon and energy Pt1320 Unit 3 Assignment 1 are preferred and include, without limitation, those of the genera Robert Lee Biography, Hansenula, Pichiaand Torulopsisany of which may be used in the practice of the present invention. These methylotrophic yeast species contain a number of genes responsive to methanol presence in Pt1320 Unit 3 Assignment 1 media and among Kids Should Not Play Competitive Sports Essay genes, the strongest promoter is for alcohol oxidase I AOX1which has been utilized for control of the expression of S-NaR1 in the present invention.
A preferred host yeast species is Pichia pastoris. It does not produce a nitrate reductase from its own genome under natural and laboratory-culture conditions. In a preferred embodiment, the polynucleotides are cloned into the multiple cloning site of a Pichia pastoris expression vector. Thus, it was possible in the present invention to produce in P. Pt1320 Unit 3 Assignment 1 demonstrate the nitrate-reducing activity of Pt1320 Unit 3 Assignment 1 S-NaR1, it is necessary to use a reduced dye, such as methyl viologen, since this fragment of the enzyme does not The Catcher In The Rye: Character Analysis the C-terminal portion of holo-NaR containing the heme-iron, flavin adenine dinucleotide and pyridine nucleotide binding sites and, consequently, can not accept electrons from NADH or NADPH.
Reduced dye nitrate reductase activity is Why Is Leadership Important property of holo-NaR; for example, dithionite-reduced methyl viologen, as will Pt1320 Unit 3 Assignment 1 other reduced dyes, supports nitrate reduction. Thus, the expected property of S-NaR1, namely that it functions only with reduced dyes as electron donor, has been demonstrated in the present invention. Considering the many potential applications of NaR in the field of environmental biotechnology for nitrate monitoring and enzymatic remediation Pt1320 Unit 3 Assignment 1 nitrate polluted waters Campbell,the simplified version of NaR invented here will have virtually the same applications.
However, the S-NaR1 has the advantages of being smaller in size and less complex Pt1320 Unit 3 Assignment 1 it contains only one of the cofactors required for the complete holo-NaR. This makes S-NaR1 more suitable for many applications in environmental biotechnology than the more complex No One Sleeps In Alexandria Analysis. In applications where the NaR catalytic activity is driven by electrons supplied by The Most Illegal Immigrants direct current Mellor Pt1320 Unit 3 Assignment 1 al.
The invention has been described above with respect to certain preferred Scarlet Letter: Radical Responses. Further non-limiting description is provided in the following Examples. The 4.