JPH05506367A - fatty acyl reductase - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] fatty acyl reductase This application is filed under USSN 07/796.256 filed on November 20, 1991. This is a continuation application and USSN 07/65 filed on February 22, 1991. USSN 07/ filed on September 27, 1991, which is a continuation in part of 9.975. This is a continuation-in-part application of No. 767, 251.

Technical field The present invention relates to plant enzymes, methods for purifying and obtaining such enzymes, and related amino acids. It relates to acids and nucleic acid sequences and methods of utilizing such compositions.

Fatty acids are organic acids having a hydrocarbon chain of about 4 to 24 carbons.

A wide variety of fatty acids are known that differ from each other in chain length, presence, number and position of double bonds. ing. Within cells, fatty acids typically exist in a covalently bonded form, The xyl moiety is called a fatty acyl group.

The chain length and degree of saturation of these molecules are often represented by the formula CX:Y. here where "X" represents the number of carbons and "Y" represents the number of double bonds. fatty acyl molecule The carbon chain always contains an even number of carbons, so we use the formula “Cxt” to represent the carbon chain length. Sometimes.

Fatty acyl groups are the main components of many lipids, and their long nonpolar hydrocarbon chains It is responsible for the water insolubility of those lipid molecules. Covalent attachment of fatty acyl groups to other factors The types of cases can be different. For example, in biosynthetic reactions, they are Depending on the elementary reaction, acyl carrier protein (AC P) or coenzyme A (CoA). In wax, The fatty acyl group is linked to the fatty alcohol via an ester linkage and the triadium Luglycerol consists of three acyl molecules linked to a glycerol molecule through an ester bond. It has a ru group.

Tria consisting mainly of long chain (having 16 or 18 carbons) fatty acyl groups A number of plants have been studied that store lipids as dilglycerol. very long Monounsaturated fatty acyl groups of the chain (having 20-24 carbons) from CI8:I It is formed by the acyl-CoA elongation process and is found in numerous plant seeds, especially Brassica (C. luciferae). Good as jojoba Simmondsia chinensis, a member of the Buxaceae family, produces a large amount of liquid. Higher plants (seed plants) in their ability to produce body waxes and store them as seed storage lipids. ) is unique among the These simple wax compounds have very long chains. It is an oxygen ester of enoic acid fatty acyl group and alcohol.

Other types of wax are also produced by some plant species. Plant epidermis or angle wax synthesis of sugar, as well as bacteria, such as Acinetobacter. r) CFixter et al. (1986) J, Gen, Microbio L, 132:3147-3157) and Micrococcus (Lloyd (1 987) Wax synthesis by Mictobios 52:29-373 and Wax synthesis by Euglena, a unicellular green algae, is It is publicly known. However, the composition and biosynthetic pathway of those waxes is Different from seed wax.

For example, in the formation of Euglena storage wax, the alcohol moiety is fatty acyl Co by NADH-dependent reduction of fatty acyl compounds catalyzed by A reductase. It has been proven that the formation of In jojoba seeds, the reaction is NADPH dependent. It is existence. The reduction of very long chain fatty acyl CoA to the corresponding alcohol is It is hypothesized that the activity of this enzyme depends on a single enzyme, and that the activity of this enzyme is (Pollard et al. (1979) Lipi ds 14:651-662; Wu et al. (1981) Lipids 16:89 7-9023, and two-stage comparison regarding the formation of plant endothelial wax. The process has been reported (Kolattu-kudy (1980) The B iochemistry of Plants (Stumpf, P., and Conn, E.E., eds.), Volume 4, pp. 571-645). NADH dependent Fatty acyl-CoA is converted to free aldehyde by the action of existing reductase, Alcohol is then converted into alcohol by the action of NADPH-dependent fatty aldehyde reductase. It is formed.

Further characterization of the enzymes responsible for the formation of wax esters in plants depends on the enzyme activity. hampered by the lack of protocols leading to the identification of sex-related polypeptides. has been done. Therefore, further research on plant fatty acyl reductase proteins is essential. Therefore, we considered a purification protocol that can identify reductase polypeptides. It is desirable to have a plan. By establishing these methods, a sufficient amount of fat syl reductase protein and the amino acid configuration of the protein. and/or fatty acyl reductase specific antibodies. body can be obtained. The resulting amino acid sequence was determined by polymerase chain reaction ( PCR) techniques or screening of cDNA or genomic libraries. useful for Alternatively, express a fatty acyl reductase protein The antibody is used to screen expression libraries to identify clones. I can be there. The clones obtained in this way were analyzed and the results showed that vegetable fat content was Nucleic acid sequences corresponding to syl reductases can be identified.

Cell-free homogenates from developing jojoba embryos contain NADPH-dependent fatty acyl Co It was reported to have A reductase activity. This activity is determined by differential centrifugation. (Pollard et al. (1979) supra: Wu et al. (1981) supra).

in the known dinucleotide-binding fold of several reductase proteins. The conservation of functional residues is explained by Karplus et al. (Science (1991) 251 : 6O-66).

Midorim with fatty acyl CoA reductase activity April 22-2, 1990 On the 4th, it was reported by Las Cruces, NM [Summary].

A 300-fold purification of the jojoba reductase protein was performed by Pu5hnik et al. It has been reported by [Summary from [verside], Ca.].

Brief description of the drawing Figure 1. Nucleic acid sequence and translated amino acid sequence of jojoba fatty acyl reductase are provided. be done.

Summary of the invention The present invention provides partially purified fatty acyl reductase proteins, The protein is active in the formation of fatty alcohols from fatty acyl substrates. Ru. The reductase of the present invention can be used for various lipids including acyl-CoA and acyl-ACP. Can be active with fatty acyl substrates. The carbon chain lengths of their substrates are different However, a given reductase can be activity with respect to acyl substrates that exhibit preference or have a wide range of carbon chain lengths. can be sexual.

Generally, the reductases of the present invention have a small chain length (both 16 to 24 carbons). length can be represented by the formula "Ctx", where "X" is a number from 8 to 12) Although active against acyl substrates, other acyl substrates were also tested and additional activity can also be discovered. In addition, the reductase protein of the present invention can be obtained. If so, operations such as those described in more detail below are also possible. Those operations are similar to other may result in the production or discovery of related reductases.

According to a first aspect, the present invention provides proteins exhibiting fatty acyl reductase enzyme activity. The present invention relates to protein preparations, such as seed plant protein preparations. such preparation The jojoba germ is fractionated to produce a microsomal membrane preparation, and this membrane preparation is The reductase protein is solubilized from the It is produced by further purification. Jojoba reductase is a very long-chain acid prefers acyl-CoA substrates, but activity with other acyl substrates is also observed, and NAD It has been shown to be PH dependent.

These procedures allow for migration as a doublet band on polyacrylamide gels. There are two major polypeptides with apparent molecular weights of approximately 54 kO and 52 kD. A partially purified reductase preparation is obtained that contains tide. Therefore, seed plants Methods of obtaining acyl reductases through purification from sources, as well as their A method of obtaining the amino acid sequence of a proteinase is provided.

Another aspect of the invention relates to nucleic acid sequences associated with reductases of the invention. Ru. Identification and identification of the amino acid sequences of the reductase proteins of the present invention and methods by which it can be obtained are described. Upon isolation of different reductase sequences for the transcription of reductase sequences and/or reductase sequences in host cells. Utilization of said structural gene sequence in a recombinant construct for the expression of tercet protein is described. Use of other nucleic acid sequences related to reductase proteins, e.g. Utilization of the 5' and 3' non-coding regions is also considered.

Yet another aspect of the invention relates to cells containing the recombinant constructs of the invention. especially , cells containing substrates preferred by jojoba reductase, such as embryonic cells of cruciferous plants. Regarding.

Moreover, as a result of expression from the recombinant constructs of the invention, It also concerns cells containing proteins and how reductase is produced in host cells. provided by law. Therefore, as a result of expression of the protein in the host cell, Also relevant to the present invention are reductase proteins that are Furthermore, the reductor of the present invention can be utilized in the production of fatty alcohols in such host cells. will be recognized.

Detailed description of the invention The fatty acyl reductase of the present invention converts fatty acyl groups into corresponding alcohols. Any sequence of amino acids, such as proteins, polypeptides, that has the activity of catalyzing reduction. peptide or peptide fragments.

A fatty acyl group is a group that is covalently bonded to a carrier, such as ACP or coenzyme A. meaning a fatty acyl group.

This enzymatic reaction involves pre-reduction of 4, which can be carried out in two steps, so that fatty acyl groups can be Reduction to alcohol may or may not require additional enzymes. Ru. In the first step, the acyl group is converted to an aldehyde, which is then converted to the corresponding acyl group. It will be returned to Lecole. Therefore, the reductase of the present invention can convert acyl to acyl. May be active throughout the four-electron reduction to alcohols or to aldehydes. catalyzes only the reduction of aldehyde to alcohol by a second enzyme. good. Evidence so far suggests that a single enzyme completes acyl-CoA to alcohol. Indicates full reduction. The fatty acyl reductase of the present invention will hereinafter be referred to as [acyl reductase]. Also called ``actase'' or ``reductase.''

The present invention provides seed plant fatty acyl reductors that convert fatty acyl groups into alcohols. Regarding ze. More particularly, the invention relates to NADPH-dependent reductases.

In addition, the plant fatty acyl reductase of the present invention has fatty acyl CoA and fatty acyl reductase. can have activity against both ACP molecules, and the observed activity can be used to It will depend on the substrate. However, fatty acid synthetase (FAS) acyl Manipulation of the CoA elongation process requires preferential activity toward very long-chain acyl-CoA substrates. desired.

According to the present invention, the seed plant fat acyl reductase protein is an integrated membrane protein. It was determined that the quality was high. In general, membrane-associated proteins are known to be soluble enzyme activity when they are separated from their normally functioning membrane environment. It is difficult to purify because it tends to lose However, it still retains enzyme activity. Obtaining solubilized seed plant fatty acyl reductase is a membrane-bound protein. It can enable a variety of uses that are not possible with proteinaceous materials.

For example, if a purified or partially purified acyl reductase protein is obtained If so, it can be immobilized and used in a reactor system to generate reduced pyridine nuclei. Fatty alcohols can be prepared in the presence of an otide regeneration system. Furthermore, Leduc Research on the tase protein led to site-directed mutagenesis studies to determine its catalytic properties. further characterization and improvement of the acyl substrate specificity or alteration of its acyl substrate specificity. Wear. Reductases with altered substrate specificity can be used in conjunction with other FAS enzymes can be found. For example, plant thioesters that prefer medium M (CI2 to C14) (see U.S. patent application Ser. No. 07/662.007) and a suitable aliase. Medium-chain alcohols using syltransferases in conjunction with modified reductases The alcohol can then be esterified to a fatty acid to form an ester. can be obtained.

Other important factors to consider when dealing with membrane-bound proteins are the It is the degree of bonding. Perimembrane proteins or membrane-integrated proteins are known. ing. Peripheral proteins are typically somewhat hydrophilic in nature and only weakly attach to membranes. It is bound and easily solubilized. In contrast, membrane-integrated proteins are found in lipid membranes. It has a highly hydrophobic region buried in There are often times when it is necessary to match.

The techniques used to solubilize integrated membrane proteins involve the preparation of appropriate membrane fractions. Including the addition of surfactants or organic solvents to the product.

If the desired protein is still functionally active (this can be measured using specific enzymatic assays) ), then other conventional purification techniques, e.g. Using precipitation, ion exchange, gel filtration and affinity chromatography I can do it.

Typically, as a first step to obtain a solubilized protein preparation, acid A microsomal membrane preparation of seed plant tissue containing reductase activity is desired. Ru. Standard microsomal membrane preparation contains whole cells, nuclei and soluble proteins. Differential centrifugation of cell-free homocyne (CFH) is used, which yields a membrane fraction that is free from oxidation.

[For example, Moor6 et al. (1987) Biological Membrane es:　A　Practical　Approach, 　pages 37-72, 　Fi See Nalay and Evans, eds. ] For oil seeds, the first centrifugation The steps typically give the pellet, upper groove and floating fat layer, and then the supernatant is further removed. Microsomal membranes can be recovered by heart separation.

Co-pending USSN No. 07/659.975 filed February 22, 1991 Therefore, the membrane fraction containing the acyl reductase protein is better than that in CFH. A protocol is described that provides a high recovery rate of reductase activity. this method A key step was the removal of the seed coat from the jojoba embryo. This is because the seed coat contains enzymes. This is because it is known to contain factors that interfere with scientific measurements. This method is professional Use a high salt solution during the first part of the tocol, that step is described below, and later Examples will also be described in more detail.

A powder is produced from the jojoba embryo sample, and the powder is mixed with high salt (3M NaCl) sucrose (0,3 M) by homogenizing in solution at a rate of 80 g of solution per 20 g of embryos Prepare homogenate. This homogenate is then filtered and ioo, ooo Centrifuge at xg for about 1 hour to obtain the pellet, upper groove and floating fat layer. remove fat layer Collect the upper groove and add 100 [OM HEPES (1) H7,5), 2mM Dialyze against IM NaCl solution containing DTT and 0.5d EDTA.

The dialysate is then dried at 100,000 Xg or preferably at 200,000 Xg Microsomal membranes with acyl-CoA reductase activity were centrifuged for approximately 1 hour at Obtain a pellet DP2 comprising:

Acyl reductase activity in microsomal membrane preparations or during further purification steps Further characterization of the optimized specific assay for acyl reductase It will become easier by developing . For example, in jojoba, it is very using long-chain acyl-CoA molecules and high salt (0.2M to 0.5M NaCl ) conditions are used. High salts are detectable acyl-CoA reduc was found to significantly increase tase activity. This assay was performed in Example 1. Please describe in detail.

Another important step in further enzyme characterization and purification is the native lipid bilayer membrane environment. It is possible that the reductase enzyme has been isolated from the The objective is to obtain solubilized reductase protein. Incorporation from lipid bilayers Desorption of type membrane proteins is typically performed using organic solvents, although in a few cases organic solvents are also used. is achieved using amphiphilic surfactants in aqueous solution. ionic and non-ionic A large number of different surfactants are available, both of their dissociative effects and their CMC, effect on enzyme activity and further purification, and solubility They differ in ease of removal from the liquid. Wide variety of surfactants and membrane proteins Quality solubilization methods are known to those skilled in the art and are described by Neugebauer (Met hods Enzymol, (1990) 182: 239-253) and Hjelmiland (Methods Enzymol, (1990) 182:253-264).

Often, the detergents used to solubilize membrane proteins are It is recognized that it inhibits the enzyme activity of the enzymes. Some surfactants exhibit broad-spectrum properties. The solubilization of jojoba acyl reductase was tested, but none of them showed inhibitory properties. It turns out that However, apparent detergent inhibition of reductase activity CH may be due to some effect other than irreversible inhibition of the enzyme. The reversibility of inhibition by APS was investigated.

The surfactant CHAPS (3-((3-chloroamidepropylene) strong inhibition by dimethylammonio-1-propanesulfonate) However, the enzyme was exposed to CHAPS on ice and then the CMC value or less Complete recovery of reductase activity was obtained when returning to the full CHAPS concentration. Yo Therefore, reductase may be irreversibly inhibited by the surfactant CHAPS. do not have. The interface provides approximately 85% of the reductase activity from microsomal membrane preparations. Protocol for solubilizing jojoba acyl reductase activity using the activator CHAPS A new rule was devised. This method is described in detail in Example 2. Similarly, other interfaces We conducted studies on the reversibility of apparent reductase inhibition by activators and determined that jojoba acyl Other components for solubilization of reductase activity or other candidate reductases specific surfactants can be identified.

Once the solubilized acyl reductase protein is obtained, the enzyme can be purified with substrate specificity. , further experiments to characterize cofactor requirements and possible inhibitors of activity. It is understood that it is possible to do For example, the jojoba acyl reductase of the present invention is A It was discovered to have a wide range of substrates including CP and CoA substrates. For example, activity towards acyl Co substrates having at least 16 carbons, and at least Activity towards acyl-CoA substrates containing 18 carbons is observed. (C15) Preferential activity towards -15-tetracocenoyl-CoA (C24:1) was observed. be done.

For example, by chromatography on immobilized reactive dyes, protein It is possible to further concentrate candidate plant acyl reductase proteins in the preparation. can. A large number of such reactive dye matrices are known, e.g. This is the case with the C1bacron BlueF3GA (Blue A) used. Book According to the invention, about 0.2M NaCl, more preferably 0.5M NaCl or More preferably, when loaded in a buffer containing 0.4M NaCl, jojoba acid reductase activity binds to such a column, while other proteins about 8 5% or more is proven to pass through or be removed by subsequent cleaning. . Furthermore, about 1. For elution from a Bloom column in a buffer containing OM Na1l It is demonstrated that more jojobaacyl activity can be recovered.

Acyl reductase activity was further determined by enriched protein preparations from Bloom columns. The product is passed through a column (often called a gel filtration column) packed with a size exclusion matrix. It is further purified by applying it to This size exclusion column is undenatured An estimate of the size of the reductase enzyme is also provided. In particular, more refined jojoba To obtain the syl reductase fraction, a column matrix for narrow size fractionation, e.g. ragel AcA34 or 5ephacryl 5100 are useful. Special It is interesting to note that during one main peak, a size exclusion column or equivalent Use to obtain a recovery rate of approximately 40-60% or more of the loaded reductase activity. Methods and buffers that can be used.

Application of reductase activity from a size exclusion column to an affinity column is resulting in further purification of the ectase protein. For example, the active fraction is approximately 0. IM Can be applied to a valmitoyl-CoA agarose column in NaCl.

Then with 15mM NADPH (cofactor for reductase enzyme from jojoba) Approximately 70% of the reductase activity can be eluted using a buffer containing .

During purification, fractions containing acyl reductase activity can be analyzed using other techniques, e.g. Can be subjected to rearacrylamide gel electrophoresis and subsequent staining. child Thus, the major polypeptide band in those fractions with reductase activity can be identified.

For example, jojoba ducts partially purified from a balmitoyl CoA agarose column. In the Tase preparation, two bands representing a polypeptide of approximately 53 kD, more specifically Two molecules representing polypeptides with apparent molecular weights of 54 kD and 52 kD A band was identified, representing over 95% of the protein in the preparation. Furthermore 5DS-PAGE analysis using various markers revealed that these reductase proteins The apparent molecular weight of the protein is more precisely 54 kD and 56 kD, or about 55 kD. Point out that it can be defined as D.

The apparent size of the native reductase enzyme was determined by size exclusion chromatography. When evaluated by It does not seem to represent two distinct subunits. Rather, a reductor enzyme activity is associated with one or both of these polypeptides. Anion exchange dye column, hexanoyl-CoA affinity column, gel filtration, heparin Additional tests including phosphorus column and 1,000-ol interaction chromatography It was not possible to obtain information on Canada.

The jojoba seeds used for this purification are collected from a diverse population of jojoba plants; These polypeptides represent closely related variants of the same enzyme, i.e. isoenzymes. It will be. using tryptic digestion and amino acid sequence analysis of two polypeptides. Thus, the 54 and 52 kD bands can be further characterized.

Recovery of substantially purified reductase protein is currently accomplished using a variety of methods. I can. For example, by running a polyacrylamide gel, proteins are carriers, such as nitrocellulose or polyvinylidene difluoride (PVDF) can be moved to. Then cut those membranes containing the identified proteins. Pieces can be obtained substantially free of other proteins. Our industry Proteins are transported from the membrane using techniques known in the art and described in the Examples below. can be further manipulated to determine their amino acid sequences.

For example, by sequencing the N-terminal amino acid region of the entire protein; by chemical cyanogen bromide digestion or by enzymatic cleavage using proteases. The amino acid sequence can be determined by preparing fragments of the desired protein. I can do it. Examples of proteases that can be used include endoproteases. nase1ysc.

Examples include gluc, AspN and trypsin. The fragments obtained in this way , can be purified and sequenced according to methods well known to those skilled in the art. .

Further characterization of the 54 kD and 52 kD candidate polypeptides, e.g. Expression of each protein in enterobacteria (E. coli) and subsequent reductor Confirmation of enzyme activity is useful. Other techniques include using antibodies specific to the candidate protein. body and proven to inhibit reductase activity in protein preparations. Immunoassays that can be used include:

Additionally, proteins associated with acyl reductase activity were determined Isolate the nucleic acid sequence derived from the amino acid sequence and determine the identity of the acyl reductase protein. of the sequence in either prokaryotic or eukaryotic host cells. It is desirable to provide for transcription and/or expression of the protein. For example, If proteins are produced at high levels in prokaryotes, such as E. coli, may be destructive or even toxic due to insertion into. Therefore, Low level expression using weak promoters may be desirable. Alternatively, membrane insertion Once the causative leader peptide is discovered, the mature reductase protein Constructs containing only those nucleic acid sequences encoding protein proteins may also be prepared. instructor The reductase protein can be produced in E. coli cells. in Escherichia coli If no reductase activity is detected in the membrane bilayer, e.g. The presence of reductase protein in E. coli cells may be The presence can be confirmed by other means, for example using antibody preparations.

As acyl reductase is a membrane-bound protein, check the reductase activity. It may be desirable to express candidate proteins in plant cells to do not have. Electroporation or bombardment of plant tissues for temporary expression can be used to useful for a purpose. ultimately produces a substrate that is recognized by the reductase enzyme reductors in plants such as members of the genus Brassica, Stable expression of the enzyme protein is desired. In this way, pharmaceuticals, cosmetics, detergents, Obtaining acyl alcohol products with applications in plastics and lubricants be able to.

The reductase nucleic acids of the invention can be genomic or cDNA, and may also be cDNA. isolated from DNA or genomic libraries or directly from isolated plant DNA. can be released. Once a protein has been isolated and/or Once the amino acid sequence is obtained, methods for isolating the gene sequence are known to those skilled in the art.

For example, an antibody can be raised against an isolated protein and used to raise an antibody against an isolated protein. The library was screened to identify plant acyl reductase proteins and or antigenic fragments thereof can be identified. Or a Synthesize oligonucleotides from amino acid sequences and use them to isolate nucleic acid sequences be able to. Oligonucleotides are useful in PCR to produce nucleic acid fragments. and then used to screen cDNA or genomic libraries. can be done. Another approach uses oligonucleotides to or Southern blot analysis and use the oligonucleotides to generate cDNA or are useful probes and hybrids when screening genomic libraries. diization conditions can be identified.

The acyl reductase nucleic acid sequences of the present invention include jojoba acyl-CoA reductase tannins. protein equivalents and those obtained from jojoba protein sequences or nucleic acid sequences. Contains arrays that can. [Equivalent] means jojoba acyl reductase zetan. encoding proteins or parts thereof, transcription of reductase in jojoba membranes, or found 5' or 3' of the coding sequence that directs transcription and translation (expression). Regulatory sequences, intronic sequences not present in cDNA, and insertion into the endoplasmic reticulum membrane may be required for the mature or processed acyl reductase enzyme. Precursor reductase protein leader or signal peptide not found in Refers to a nucleic acid sequence, either DNA or RNA, including sequences.

A jojoba sequence or a sequence “obtainable” from a protein is a jojoba acyl Desired fatty acid reductase that can be synthesized from the reductase amino acid sequence Any nucleic acid sequence related to a protein or identified in a different organism and for the jojobareductase nucleic acid sequence or jojobareductase protein. refers to any nucleic acid sequence isolated using an antibody prepared as a probe. .

Thus, jojoba sequences can be used for nucleic acid hybridization or antigenic methods. the sequence of another acyl reductase isolated from the desired organism by either It can similarly be used to isolate further acyl reductases. jojoba Such a reductase derived from a seed plant reductase isolated by Reductase is likewise considered here as ``obtainable J.''

Techniques and plasmids or viruses known to those skilled in the art for the isolation of nucleic acid sequences Vectors can be used to prepare cDNA or genomic libraries.

Useful nucleic acid hybridizers that can be used to screen for desired sequences cation and immunological methods are known to those skilled in the art, eg Maniatis (Molecular’ Cloning: A Laboratory Ma nual, 2nd edition (1989) Co1d Spring Harbor La bo-ratory, Co1d Spring Harbor, New Y ork).

Typically, the sequence that can be obtained from the use of a nucleic acid probe is the target sequence and the sequence of interest. 60-70% between a given sequence encoding an acyl reductase enzyme will show sequence identity. However, 50-60% shows how low sequence identity It is also possible to obtain long sequences with Nucleic acid probes are long fragments of nucleic acid sequences or shorter oligonucleotide probes. long nucleic acid fragments (larger than about 100 bp) when used as a probe. 20-50% deviation from the used sequence (i.e. 50-80% sequence homology) can be screened with low stringency to obtain sequences from targeted target samples. Ru. The oligonucleotide probe is a complete oligonucleotide encoding the acyl reductase enzyme. may be significantly shorter than a typical nucleic acid sequence, but at least about 10, preferably at least It should be about 15, more preferably at least about 20 nucleotides. long A higher degree of sequence identity is desired when shorter regions are used. Okay In order to identify enzyme active sites with high amino acid sequence identity and detect homologous genes. It is desirable to design oligonucleotide probes for this purpose.

Whether related genes can be isolated by hybridization with a given sequence To determine whether the sequence is Make it. Other methods are also available. Add the labeled probe to the hybridization solution. containing the desired nucleic acids, either Northern or Southern blots. filter (to screen desired sources for homology), or a filter containing the cDNA or genomic clone to be cleaned; Incubate together. Hybridization and washing conditions should be changes to optimize hybridization of the probe to the sequence of I can do it. Lower temperatures and higher salt concentrations reduce hybridization of more distantly related sequences. (low austerity). Background high under low stringency conditions Detection of specifically hybridizing sequences when hybridization is a concern Increase the temperature during hybridization or wash steps to improve and/or the salt concentration can be reduced. hybridize tion and washing temperature as described by Be1z et al. (Methods in Enzymo! Probe as discussed in Ogy (1983) Gallihon 266-285:l. can be adjusted based on the estimated melting temperature of the tube.

Useful probes and appropriate hybridization and wash conditions as described above. After identification, cDNA or genomic DNA is isolated using labeled sequences and optimal conditions. Screen your library. First, the library was placed on a solid agar medium. smear the DNA onto a suitable membrane, usually a nitrocellulose or nylon filter. - move upward to -. Then attach those filters to the probe and hack as described above. Hybridize, wash, and identify clones containing the relevant sequences.

For immunological screening, inject purified protein into rabbits or mice. Antibodies against jojoba acyl reductase can be prepared by Ru. Methods for preparing such antibodies are known to those skilled in the art. monoclonal or poly Although either clonal antibodies can be produced, typically polyclonal Antibodies are more useful for gene isolation.

To screen for desired plant species, Western analysis was performed to Crude extraction of desired plant species for relevant proteins that cross-react with antibodies against ductase Determines its existence in things. This is applied onto a membrane, usually a nitrocellulose membrane. Immobilization of plant extracted proteins, electrophoresis, and incubation with antibodies This is achieved by Antibody/protein complexes on nitrocellulose filter Various systems are available for detection, including Radiolabels and second antibody/enzyme conjugate systems are included. some available systems is 0berfelder (Focus (1989) BRL㇠ifeTec Hnologjes, Inc. 11:1-5). cross anti Once responsiveness is observed, an expression library representing the desired plant species can be screened. The genes encoding the relevant proteins are isolated by expression library - λgtll as described by Maniatis et al. (supra) can be produced in a variety of commercially available vectors, including:

using DNA hybridization or immunoscreening techniques as described above. The clones identified are then purified and the DNA isolated and analyzed using known techniques. Ru. This ensures that the clone encodes the relevant acyl reductase protein. Check that. those reductors in the same way as with jojoba reductase. can be used to obtain other seed plant fat acyl reductases.

The acyl reducts of the present invention can be produced using site-directed mutagenesis or PCR detection techniques. The enzyme nucleic acid sequence can be modified or synthesized, or used when producing the synthetic nucleic acid sequence. It will be recognized by those skilled in the art that column changes can be made. those strange Altered sequences are also considered acyl reductase nucleic acid sequences of the present invention. example For example, changing codon fluctuations so that the nucleic acid sequences encode the same amino acid sequence. Alternatively, codons can be changed to result in conservative amino acid substitutions. can be done. In either case, the peptide or protein has the desired enzymatic activity. and are therefore considered part of this invention.

The nucleic acid sequence of the acyl reductase enzyme of the present invention can be obtained from genomic DNA, cDNA, mR DNA or RNA sequences derived from NA, or in whole or in part May be separated and synthesized. Gene sequences can be obtained, for example, by isolating genomic DNA from an appropriate source. , and then use polymerase chain reaction (PCR) to amplify and clone the sequence of interest. Cloning can be performed by Or, especially plants Synthesize the gene sequence completely or in part if it is desired to provide the sequence be able to. All or part of the desired structural gene (reductase protein) the part of the gene that it encodes) can be synthesized using codons preferred by a particular host. can. Host-preferred codons are e.g. proteins expressed in the desired host bull. It can be determined from the most frequently used codons.

Nucleic acid sequences related to acyl reductase proteins may find numerous uses. cormorant. For example, a recombinant construct can be prepared and the recombinant construct acyl reductase protein in the host cell I will prepare for the manifestation. Depending on the intended use, the composition may contain the entire reductase or can contain an array encoding that part. For example, the weight of the reductase Key regions, such as active sites, can be identified. required for the desired reductase activity. Prepare another construct containing only the portion of the reductase sequence that encodes the essential amino acids You can also.

Expression in host cells containing the preferred substrates of acyl reductase proteins can provide for the production of fatty acyl alcohols from fatty acyl substrates. Re Useful systems for expressing ductase proteins include prokaryotic cells, such as E. coli, mother cells, and plant cells, with vascular and non-vascular plant cells being preferred. It is a good host. In this way, reductase protein can be produced. Wear. In addition, using site-directed mutagenesis of the coding sequence, reductase The effect of specific mutations on protein reactivity can be investigated.

Further, provision is made for transcription of complementary sequences of the acyl reductase coding sequence or fragments thereof. Antisense constructs can be prepared. Thus, live in the target host organism. The amount of reductase protein produced can be reduced.

DNA sequences encoding the acyl reductases of the invention can be derived from foreign DNA in a variety of ways. Can be combined with A array. “Foreign JDNA sequences are naturally occurring means any DNA sequence not found linked to a is a combination of DNA sequences from the same organism that are not found linked together in nature. can be mentioned. For example, a signal peptide may be encoded in the reductase sequence of the invention. It is desirable to concatenate the arrays. Thus, fatty acyl substrates, especially fatty The reductase can be directed to the chloroplast where acyl ACP is available. Ru.

The DNA sequence encoding the acyl reductase of the present invention is usually It can be used in conjunction with all or part of the linked gene sequence. Its structure In the 5′ to 3′ transcript, the reductase-encoding DNA sequence transcription initiation regulatory region that can promote transcription and translation in the host cell in the direction , a recombinant construct having a nucleic acid sequence encoding a reductase and a transcription termination region be combined inside.

Depending on the host, the regulatory regions will be different, but may be viral, plasmid or Contains regulatory regions from chromoplast genes, etc. in prokaryotic or eukaryotic microorganisms, especially in unicellular hosts A wide variety of constitutive or regulatable promoters can be used for expression in . Expression in microorganisms can provide a ready source of plant enzymes. listed Among the transcription initiation regions in bacterial and yeast hosts, such as E. coli (E. i) Gene-derived genes such as Dase, T7 polymerase, Tryptophan E, etc. It is an area.

For the most part, the recombinant constructs are prepared in plants for the production of acyl reductase. contains functional regulatory regions. Encoding a plant reductase or a functional fragment thereof The open reading frame will be linked to the 5' end of the transcription initiation regulatory region. Translation start area The translation initiation region may be the 5' region of the reductase cDNA sequence. from a non-coding region or from a translation initiation region originally linked to the transcription initiation region of the construct. can be provided. Generally, the combination of transcriptional and translational regulatory regions is - is called. constitutive or regulated expression of a wide variety of structural genes in plants; For example, a number of promoters are available that provide for inducible expression.

of sequences known to be useful in providing for constitutive gene expression in plants. Some of them are related to Agrobacterium genes. regulatory regions such as tubalin synthase (Nos), mannopine synthase ( Mas) or octopine synthase (Ocs) genes, as well as viral genes. The region encoding the expression of, for example, the cauliflower mosaic virus (CaMV) There are 35S and 19s regions. As used herein, the term "constitutive" Point out that genes that do not necessarily contain genes are expressed at the same level in all cell types. Although certain deformities are often found in abundance, rather than It is shown that the gene is expressed in a wide range of cell types. Other useful transcription initiation regions The small distribution of RUBISCO in certain tissues or under certain growth conditions This is the transcription initiation region from the block unit, etc.

In embodiments where expression of the acyl reductase protein in a plant host is desired, complete It may be desirable to use all or part of the entire plant acyl reductase gene. i.e. the 5' upstream non-coding region (promoter), the structural gene sequence and the 3' downstream You can use free code areas. Different promoters, e.g. A promoter that is native to the main or a modified promoter, i.e. It has a transcription initiation region derived from one gene source and a translation initiation region derived from another gene source. promoters, or enhanced promoters, e.g. dual 35S CaMV promoters. If desired, the arrays can be concatenated together using standard techniques. can be done.

Applications from another gene whose 5' upstream non-coding region is regulated during seed maturation In this case, those that are predominantly expressed in the liver tissue of plants, such as ACP or nabin-derived A conventional transcription initiation regulatory region is desired. Such [seed-specific promoters] , U.S. Patent Application No. 07/147.781, filed January 25, 1988 (199 U.S. Patent Application No. 07/742.834, filed Aug. 8, 1999), and “Nov. el 5equences Preferentia11ymixpressed In Early 5eed Development and Method 1990 3 with the name of the invention “ds Re1ated Thereto” Technology of U.S. patent application Ser. No. 07/494.722 filed on or about May 16th. may be obtained and used in accordance with. All those concurrently pending applications are for reference. Incorporated herein as . Transcription initiation region predominantly expressed in seed tissues area to minimize destructive or adverse effects of the gene product on other plant parts. Therefore, it is considered desirable for fatty alcohol production.

Transcription termination regulatory regions can also be provided in the recombinant constructs of the invention. transcription termination The region can be derived from a DNA sequence encoding a plant acyl reductase or from another gene source. Convenient transcription termination regions derived from, especially transcription termination regions originally associated with transcription initiation regions It can be provided by The transcription termination region is typically derived from at least about 0.5 kb, preferably about 1 to 3 kb 3' of the structural gene for will contain an array.

Plant expression constructs that nurture plant acyl reductases as DNA sequences of interest for expression. are found in various plants, especially in very long-chain fatty acyl-CoA molecules (particularly in rapeseed). used in plants that produce erucic acid (mutants), such as Brassica (Brassica). can be used. Other plants of interest have desirable substrates, e.g. medium-chain or long-chain produces fatty acyl molecules, such as rapeseed (Ca nola variety), sunflower, safflower, cotton, crape myrtle (Cuphea), large Examples include beans, peanuts, coconut and oil palm, and corn. Not limited to these. Depending on the method of introducing the DNA expression construct into the host cell , other DNA sequences may be required. Importantly, the present invention Applicable to cotyledons and novel and/or improved transformation and regeneration This is something that could be easily applied to technology.

The method of transformation is not critical to the invention. Various methods of plant transformation are currently available. Currently available. More novel methods are available for transforming grains. and they can be applied directly according to this description. For example, agrobaku Many plant species that are naturally susceptible to infection by Agrobacterium are , binary-vector method of Agrobacterium-mediated transformation or tripartite hybridization can be successfully transformed via . of the nucleic acid sequence into the host plant cell. Other sequences that are useful for preparing for transport include plant pathogenic viruses or plant translocation agents. It can be induced from the child. In addition, traits of various monocot and dicot species Microinjection, DNA particle bombardment, electroporation in preparation for transformation technology has been developed.

When developing a recombinant construct, the various components of the construct or fragments thereof are typically inserted into a convenient vector capable of replicating in a bacterial host, such as E. coli. It will be. There are numerous vectors described in the literature. Each cloning Afterwards, the plasmid is isolated and subjected to further manipulations, e.g. restriction, insertion of new fragments, ligation, deletion. A desired sequence of components can be assembled by deletion, insertion, excision, etc. one end structure Once the product is completed, it is then subjected to further manipulation depending on the method of host cell transformation. can be inserted into a suitable vector for

The recombinant construct usually contains the regulatory sequences necessary for expression in the host and Structural genes will be included to provide for selection of transformed cells. The gene is a cytopathic Resistance to harmful factors, such as antibiotics, heavy metals, toxins, etc. Provision can be made for supplementation to provide nutritional properties, viral immunity, etc. Similarly, color Enzymes, such as GUS, which provide for the production of compounds that can be identified by changes in Or there is a gene encoding an enzyme, such as luciferase, that prepares for the production of luminescence. It is for use. The selection of transformed tissues depends on the various host species into which the expression construct is introduced. One or more markers can be used for selection or detection, in which case Different selection conditions are used for hosts.

Using Agrobacterium for plant transformation nucleic acid sequences bordered on one or both ends by T-DNA, especially in the left and right border regions. It would be desirable to have an area, more particularly at least a right border area. So These border regions may also be useful when using other transformation methods.

Agrobacterium (Agrobacterium) or Rhizogenes (R hizogenesis) sequence present in the host when used for plant transformation. T]- or R1- due to homologous recombination with T-DNA on the plasmid. Vectors that can be introduced into the Ctellium host may also be used. for recombinant T-DNA-containing Ti- or Ri-plasmids may be armed (causing lump formation). can be disarmed (can induce lump formation) or disarmed (can cause lump formation). ). The latter is a trans-acting agent required for the transport of DNA into the plant host cell. Functional complementation of the vir genes encoding sex factors can be achieved in transformed Agrobacterium hosts. It is permissible as long as it exists in the Lord. Using armed Agrobacterium strains , yielding a mixture of normal plant cells, some of which contain the desired nucleic acid sequence. The plant cells are capable of tumor formation due to the presence of oncogenic genes. can. Cells containing the desired nucleic acid sequence but lacking the oncogene undergo normal transformation. It is possible to choose from mixtures such that replacement plants are obtained.

Preferably using Agrobacterium as a vehicle to transform host plant cells In the method, the expression or transcription construct flanked by T-DNA border regions comprises: E., a broad host vector capable of replicating in E. coli and Agrobacterium It will be inserted inside. Such broad host vectors have been described in the literature. There is. pRK2 or its derivatives are commonly used. For example, Ditta (Proc.

Natl, Acad, Sci, USA (+980) 77:7347- 7351) and EPA O120515. These are the books for reference Incorporated into the specification. Alternatively, the sequence to be expressed in plant cells, Separate replication sequences (one that stabilizes the vector in E. coli, the other that stabilizes the vector in E. coli; Stabilizing the vector in Globacterium) can be done. For example, McBride and Summerfelt (Pl ant Mo1. Biol, (1990) Kai: 269-276). and. In the said literature, pRiHRI CJouanin et al., 1Jo1. Gen , Genet, (1985) 201: 370-374) We have prepared for adding stability to plant expression vectors in Agrobacterium host cells. It is growing.

Using vectors such as those mentioned above that can replicate in Agrobacterium is preferable. This method does not require plasmid recombination and the host The Globacterium vir region is responsible for the import of T-DNA border sequences into plant host cells. can supply the trans-acting factors necessary for transport.

For transformation of Brassica cells, for example, Agrobacterium can be used. One such method is Radke et al. eor, Appt, Genet, (1988) 75:685-694) It is described more.

Having thus far described the invention generally, it will now be understood by reference to the following examples. Therefore, it will be easier to understand. The examples below are provided for illustrative purposes only. They are not intended to limit the invention unless otherwise specified.

Acyl-CoA levels in microsomal exchange preparations or solubilized protein preparations A method for assaying ductase activity is described.

A. Radioactivity detection material [14C] Potassium cyanide is reacted with the corresponding alkyl mesylate, and then By base hydrolysis of alkyl nitriles to free fatty acids, long-chain (1-”C ) fatty acid (specific activity 51-56 Ci 1 mol), i.e. 11-cis-eicosenoic acid, Prepare 13-cis-tocosenoic acid and 15-cis-tetracosenoic acid. A These fatty acids are converted into methyl esters using esteric diazomethane for preparation. Purify by silver nitrate thin layer chromatography (TLC). The fatty acid methyl ester hydrolyze the terres back to free fatty acids. Evaluate radiochemical purity using three TLC methods Value: normal phase silica TLC1 silver nitrate TLC1 and C18 reverse phase TLC, their The radiochemical purity determined by the method was 92-98%. Young and Lynen (J, Biol, Chem, (1969) 24 Rainbow 377) By the method of 1 mole of 10 Ci from the corresponding long M (1-" Prepare a long chain (1-”C) acyl-CoA. Other (1-”C) acyl-CoAs , e.g. Purchased from Linton Heights, [L]. (1-14C) Sadekanal uses the method of Pletcher and Tate (Tet, Lett , (1978) 1601-1602) according to the microscale modification of (1 -”C) Prepared by dichromate oxidation of hexadecane-1-ol. Product was purified by preparative silica TLC and incubated in hexane at -70°C until use. Store as a solution.

■, Assay I: 20 μM (1-14C) acyl-CoA (usually Tetraco Senoyl CoA, specific activity 2-5 Ci 1 mol) was added to the assay sample and 2mM By incubating with NADPH in a volume of 0.25 ml, Measure reductase activity in microsomal membrane preparations.

The incubation mixture further contained 10% (w/v) glycerol, 1mM Contains DTT, 50mM HEPES (4-(2-hydroxyethylcou-1- piperazine ethanesulfonic acid) (HEPES referred to herein and hereinafter) (added from a 1M stock solution adjusted to pH 7.5).

Start the assay by adding acyl-CoA substrate and incubate for 1 hour at 30°C. vation. Place the assay tube on ice and immediately add 0.25 ml of isopropylene. Perform the assay by adding Ropatool:acetic acid (5:1.v/v). Stop. Unlabeled wax ester (0,1■) and oleyl alcohol ( 0.1■) is added as a carrier.

Hara and Radin (Anal, Biochem, (1978) 90:420) to extract (“C) lipids. . Add 61d hexane/isopropanol (3:2. v/v). The sample was vortexed and diluted with an aqueous sodium sulfate solution (5 , 5%, W/V) and vortex the sample again.

2. Assay 2: 20 μM (1-”C)acyl-CoA (usually tetracose Noyl-CoA, specific activity 2-5 Ci 1 mol) was added to the assay sample and 2mM N By incubating with ADPH in a volume of 0.25- Measure reductase activity in rosome membrane preparations.

The incubation mixture was further supplemented with 10% (w/v) glycerol, 1 ml J Contains DTT, 50d HEPES (4-C2-hydroxyethylcou-1- piperazine ethanesulfonic acid) (HEPES referred to herein and hereinafter) (added from a 1M stock solution adjusted to pH 7.5). too and acyl-CoA further present in the original preparation: alcohol acyl transfer desired to inhibit reductase activity (which depletes the reductase reaction product). If so, include 0.3% w/v CHAPS in the assay mixture. This C HAPS concentration has minimal effect on reductase enzymes but Completely inhibits the spherase reaction, thus simplifying the quantification of reductase activity .

Start the assay by adding acyl-CoA substrate and incubate for 1 hour at 30°C. vation. Place the assay tube on ice and immediately add 0.25 ml of isopropylene. Perform the assay by adding Ropatool:acetic acid (5:1.v/v). Stop. Unlabeled wax ester (25 μg), oleyl alcohol (50 μg) μg) and oleic acid (50 μg) are added as carrier. )Iara and Radin (Anal, Biochem, (1978) 9042 [14C] lipids are extracted using a scale-down protocol of [0]. stopped Add 4-hexane/isopropanol (3:2.v/v) to the assay solution. Add. The sample was vortexed and diluted with 2rnI aqueous sodium sulfate solution (6,7% , W/V) and vortex the sample again to remove the solubilized reductase activity. In order to assay the is necessary. The assay buffer for the solubilized reductase assay is As shown above for the microsomal membrane preparation assay, except that: accompanied by changes.

a. Add NaCl to a final concentration of 0.3-0.5M.

b, ~1+nM EDTA included. and C, the enzyme sample to be assayed. (contains 0475% CHAPS) to 50.3% (CHAPS The CMC of S is ~0.5%).

D. Analysis of assay products Microsomal membrane preparation reductase assay or solubilized reductase assay Two protocols were developed to analyze either product. below One protocol, described as a "detailed assay," is time consuming but also gives more advanced quantitative results. Others listed below as “rapid assays” This protocol provides a measure of reductase activity and is faster and more convenient. However, the quantitative nature is low.

1. Detailed assay: After adding sodium sulfate and vortexing the sample, the above The organic phase was removed and the lower aqueous phase was diluted with 4-hexane/isopropanol (7:2.v /v). The organic phases are pooled and evaporated to dryness under nitrogen. liquid residue resuspended in a small volume of hebutane and an aliquot placed in a liquid scintillation cow. The radioactivity is analyzed by testing. The remainder of the sample was subjected to TLC analysis of Rinji species, or Can be used for derivatization to cleave wax esters, thereby producing The amount of total alcohol produced can be given.

For lipid class analysis, samples were applied to a silica gel TLC plate and the plate The solution is diluted with hexane/diethyl ether/acetic acid (e.g. 80:20:1 or 70: 30:1 V/V/V). Roughly wax esters (riggers) (similar to the microsomal membrane preparation assay), free fatty acids, fatty acids The distribution of radioactivity among the lipid classes, alcohol and polar lipids of origin, was determined by AMB [ S Radiometric Image Processing System (AMB [S System Inc., San Measured using Diego, CA). If necessary, specify individual lipid classes. It can be recovered from the TLC plate and used for further analysis.

For the cleavage of wax esters, Pina et al. (LiPids (1987) 22 :358-361] based on the Grignard derivatization protocol. protocol. The sample was mixed with 200 μg of carrier wax ester. Both are dried in Teflon-coated glass test tubes with screw caps.

Dry diethyl ether (0.4 ml), ethyl acetate (3 μl) and diethyl ether 3M ethylmagnesium bromide (0,IrILl) in ether is added sequentially. trial The mixture was vortexed and allowed to stand at room temperature for at least 2 hours, after which it was added to a die saturated solution of water. Decompose excess reagent by carefully adding thyl ether. IM HCI and Hexa 2- of each sample and vortex the tube. Wash the upper organic phase with water 2x2 ml') and evaporated to dryness in the presence of 50-100 μl of ethanol.

Resuspend the sample in 50-100 μl of hexane and apply to the TLC plate.

Both normal phase and reverse phase TLC systems were used for analysis. Normal phase TLC is silica TLC pre Hexane/diethyl ether/acetic acid (70:30+2 v/v/ Expand in v). The reverse phase system uses a C18 plate and is developed in methanol.

2. Rapid analysis: After addition of sodium sulfate and vortexing of the sample, a known proportion of The organic phase is removed and counted by liquid scintillation counting.

Use this calculation to estimate the total counts in the organic phase. Take another part of the organic phase removed, dried under nitrogen, redissolved in heptane, and further assayed. Scan as described. In this way, it is incorporated into alcohol. A percentage of the total runt is determined.

Example 2 - Characterization of jojoba acyl-CoA reductase Development of reductase activity A method of obtaining a jojoba protein preparation and the results of studies on the activity of this enzyme are presented. available.

A. Seed formation and acyl-CoA reductase activity profile for California At Davis, embryogenesis was followed over two cycles in five types of plants. Embryo live weight and It was observed that the dry weight increased at a fairly uniform rate from about 80 days to about 130 days. It was done. Lipid extraction begins when the embryo's live weight reaches approximately 300 mm (approximately 80 days). It has been shown that the ratio of heavy to dry weight reaches a maximum level of 50%.

Acyl-CoA reductase activity was determined in developing embryos as described in Example 1. was measured. Since the jojoba seed coat was determined to be the source of the inhibitory factor, the seed coat was removed. After removal, the embryos were frozen in liquid nitrogen and stored at -70°C.

Acyl-CoA radar when measured either in cell-free homogenates or in membrane fractions The developmental profile of Cactase activity shows a peak of approximately 1158 days after flowering. We note a massive induction of ectase activity. Therefore, embryos for enzymological studies should be prepared after flowering. For about 90 to 110 days, reductase activity is high and lipid deposition reaches its maximum level. The seeds were harvested at a time when the seed coat had not yet reached bell and the seed coat could be easily removed. reductor The maximum rate of increase in enzyme activity is observed between 80 and 90 days after flowering. Therefore, Embryos for cDNA library construction are probably The seeds were harvested 80 and 90 days after flowering, when the degree of ripeness would be at its maximum.

Similarly, levels of mRNA encoding acyl-CoA reductase decreased during this period. would be expected to be the maximum.

B. Fractionation research Early attempts to fractionate jojoba embryo samples revealed that the fat layer resulting from centrifugation, the supernatant and the variable distribution of reductase activity in the precipitate fraction was obtained. Potentially active A number of treatments affect sex distribution, such as sonication, flotation gradients, and extraction slowness. The addition of various agents to the solution was tested. The salt formulation in the extraction buffer was 100.0 Maximize recovery of glue gauze activity in the upper groove fraction by centrifugation for 1 hour at 0.00 improved. Extraction buffer 3M NaCl, 0.3M'z a sugar, 100 mM HEPES, 2mM DTT, and 1mM as protease inhibitor M EDTA, 0.7 ug/i leupeptin, 0.5 μg/- pepsta Chin and 17 μg/- of phenylmethylsulfonyl fluoride (PMSF) It consists of

C1 microsomal membrane preparation After dialysis by centrifugation at 100,000 x g for 1 hour or by ammonium sulfate precipitation. Obtaining particles with high levels of reductase activity from the supernatant due to the lees Can be done. The dialysis method is described in detail in Example 3. A substance with reductase activity Further analysis of these particles, e.g. density gradient centrifugation, gel permeation chromatography, and protein/phospholipid analysis establish that the particles represent a membrane fraction. Ru. This membrane preparation also has high cytochrome C reductase activity, which is associated with vesicle Body (ER) It! used as a marker. Those studies are based on reductase Establish that the protein is membrane associated.

For the ammonium sulfate fractionation, 1 Obtain 00.000Xg supernatant. Same volume of ammonium sulfate solution (33.2g/ Ammonium sulfate concentration (while stirring) slowly add 10 (7) to the supernatant fraction. to 30%. This concentration is the concentration that will effectively precipitate the reductase enzyme. degree. After stirring for an additional 30 minutes, the supernatant was incubated at 26,000 x g for 30 minutes. After centrifugation, the resulting pellet was mixed with 25mM HEPES, IM NaCl, 1mM DTT, 0.1mM P! The first supernatant fraction Sl using a solution consisting of jsF Resuspend in a volume of 1710. This suspension at 100,000×g Centrifuge for 1 hour, and add 25mM HEPES to the resulting pellet.

Resuspend in 10% glycerol (again in 1/10 of the Sl volume). child Microsomal vectors were washed by centrifugation of the suspension at 100.000 x g. Let P4 was obtained, which was mixed with 25mM HEPES of 1/2o of Sl volume, 10 % glycerol to give a protein concentration of approximately 3-4 µ/-. later Freeze the finished coat at -70°C for use.

Jojoba reductase was assembled using the Triton X114 phase fractionation method. whether it is an integral membrane protein or is more weakly bound to MH (more closely related). aqueous protein). This technique essentially consists of membranes and 1% Tr on ice. iton X114 and then lower the temperature of the mixture to those conditions. including raising the temperature above the cloudy point of the surfactant under conditions (the cloudy point is not This is the temperature at which large micelles spontaneously form, and 1% Triton ~20°C). Upon centrifugation, two separate phases are observed, the bottom is a surfactant-rich phase, and the upper one is a surfactant-poor phase (referred to here as the aqueous phase). ). Integrated membrane proteins are predominantly partitioned into the surfactant-rich phase. , it has been shown that more hydrophilic proteins are recovered in the aqueous phase. jojoba When membrane preparations are subjected to this Triton X114 phase fractionation protocol, reductase reductase activity is present in the surfactant-rich phase, and there is no reductase activity in the aqueous phase. Not detected. This proves that the reductase enzyme is an original protein. It is based on

E. Further characterization of the reductase enzyme. used for further characterization of the enzyme. The reductase enzyme has a pH range of 5 to 9. It was shown to be active. Characterization experiments approximate the estimated physiological pH of the cytoplasm. The test was carried out at a pH of 7.5.

■, Effect of salt: - For basic salts, use a standard concentration of 0.5M to Their effects on enzyme activity were investigated. Salts containing divalent cations or anions was investigated at 0.167M (0.5M - giving the same ionic strength as the base salt). 0 ．． Up to 15-fold stimulation is observed with the addition of 5M NaCl. -Valence and divalence, etc. salts (e.g. LiC1, KCI, MgC1z, CaClt and Na25O 4) was also found to stimulate reductase activity, but compared to NaCl stimulation. In comparison, the amount was generally small. KSCN, a strong chaotropic salt, and and Na5CN provide no or marginal stimulation of reductase activity. Gave.

2. Other effectors: Dithiothreitol (DTT) has reductase activity Ethylenediaminetetraacetic acid (EDT) was found to be irritating but not essential. A) gave some stimulation and its optimal concentration was 2.5mM. low (0,0 2 to 0.075 ■/d’) Small in BSA (bovine serum albumin) concentration However, at BSA concentrations of 0.2/- or higher, inhibition of activity was observed. observed.

Acyl-CoA reductase has NADPH-specific activity (Pollard et al., supra). This confirmed the earlier observation that . NADH dependence above background No activity was measured (2% of NADPH-dependent activity).

In addition, both CoA and NaDP, which are the water-soluble final products of the reductase reaction, also gave significant inhibition of activity (at millimolar concentrations), while NADH and NAD” It had a marginal effect on activity.

3. Substrate specificity: thousand onysters, acyl ACP and fatty acids of various chain lengths Acyl-CoA was compared as a substrate for reductase enzymes.

Tetracosenoyl-CoA (24:I-CoA) substrate is a strong group at high concentration. Tests were performed at a substrate concentration of 10 MM as it showed inhibition of quality. in those assays. The NaCl concentration used is 0.15M. The results of the substrate specificity experiments are provided in Table 1 below.

Table 1: Acyl specificity of reductase 12:O<0.01<Q, 15 24:1 19.9 Since tetrafucenoyl-CoA had the highest substrate activity among those tested, Use this for reductase assays in further enzyme purification and characterization experiments. . Valmitoyl-CoA (C16:O-CoA) and Valmitoyl of interest -ACP (C16:0-ACP) was used as a substrate for direct comparison. Although the activity against Ru-CoA was slightly above background, , the activity against valmitoyl-ACP was high. Previously, stearoyl-AC It has been shown that P (Cl8:0-ACP) has activity as a substrate ( Pollard et al., supra).

It is also noteworthy that valmitoyl-CoA has a poor resistance to reductase enzymes. Although unlabeled balmitoyl-CoA (0-30 μM) and (1-14C)tetracosenoyl-CoA (20MM). In competitive inhibition experiments, reductase activity against tetracosenoyl-CoA was 50% inhibition occurred at 5 μM valmitoylu-CoA. Yo Thus, although valmitoyl-CoA is a poor substrate under assay conditions, It is an effective inhibitor.

4. Reductase inhibitor assay: Other types of reductase proteins Some known inhibitors were evaluated for their effects on jojobaacyl-CoA reductase activity. The fruit was tested. HMG-CoA reductase (3-hydroxy-3-methyl Mevinolin, a potent inhibitor of luglutaryl-coenzyme A reductase), Compared to the inhibitory concentration for MG-CoA reductase (approximately 1 nM), the ratio It was effective only at relatively high concentrations (100 μM). Cerulinen is β-keto It is well known that jojoba acyl co-binds with acyl thioester synthase. It has no strong inhibitory effect on CoA reductase.

Sulfhydryl blockers were also screened for their effects on reductase activity. did. N-ethylmaleimide was found to strongly inhibit the activity, but p-ethylmaleimide Hydroxymercrylbenzoate has some inhibitory effect, and iodoacetate Amide had no effect at all. This evidence suggests that acyl-CoA reductases are The essential sulfhydryl group exhibits considerable selectivity for sulfhydryl blocking reagents. This leads to the conclusion that it exists.

Example 3 - Purification of acyl-CoA reductase Jojoba with reductase activity Isolation of membrane preparations, solubilization of reductase activity and purification of reductase proteins. Describe methods that can be used for manufacturing.

A, microsomal membrane preparation Post-anthesis 90-11 as assessed by measuring embryo moisture (45-70%) Harvest jojoba embryos on day 08. Remove the outer shell and seed coat and quickly remove the cotyledons in liquid nitrogen. Freeze and store at -70''C for further use.

For initial protein preparation, frozen embryos were incubated at liquid nitrogen temperature in liR milk. Grind by rubbing with a bowl and pestle. In a typical experiment, 70 g of embryos are processed. Ru.

Add the powder to the following high salt solution at the rate of 280 rnl of solution per 70 g of embryos = 31J NaCl, 0, 31J sucrose, 100d HEPES, 2a+M DTT and protease inhibitor 1ml 1J EDTA, 0.7μg/- Leupeptin, 0.5 μg/rnl pepstatin and 17 Mg/- PM in the buffer for about 30 seconds using an SFo Polytron tissue homogenizer. Create cell-free homogenate (CF) by dispersing powdered embryos in . This homogenate was coated with three layers of Miracloth (CalBioChem, LaJolla, CA) and the filtrate at 100,000 x g for 1 hour. Centrifuge for a while.

The resulting sample consists of pellet, supernatant and floating fat layer. remove the fat layer, The superior groove fraction was collected and IM NaCl, 100mM HEPES, 2mM DTT and and 1 mAJ EDTA overnight (with 3 changes of buffer). ). The dialysate was centrifuged at 200,000 x g for 1 hour. give. The pellet was infused with 25mM H at approximately 1/20 of the original CFH volume. EPES (pH 7,5), 10% (w/v) glycerol, 1mM E Resuspend in DTA and 0.5 M NaCl to give microsomal membrane preparation. .

Activity is assayed as described in Example 1. Acyl-CoA reductase cycle The yield is estimated at approximately 30% of the original activity in the cell-free homogenate. This preparation The acyl-CoA reductase activity in it is stable when stored at -70°C.

Solubilize the B6 reductase protein to give a final concentration of 2% (w/v). In addition, solid CHAPS (3-((3-chloroamide protein) was added to the microsomal membrane preparation. dimethylammonio]-1-propanesulfonate). sample Incubate on ice with slow shaking for approximately 1 hour, then add 25mM H EPES (pH 7.5), 10% glycerol, 0.34M NaCl, 1m Dilute with M EDTA to bring the CHAPS concentration to 0.75% and the NaCl concentration to approx. Lower it to 0.4M. The sample was then centrifuged at 200,000 xg for 1 hour, Harvest the supernatant and assay for reductase activity as described in Example 1. . Typically, 85% of reductase activity from microsomal membrane preparations is present in the supernatant fraction. collected in minutes. Solubilized reductase activity is stable when stored at -70°C. It is.

C1 Blue A Column Chromatography Column with a bed volume of approximately 25- 1,8X to 10an), and add Blue A (Cibacron Blu e F3GA; Am1con Division, W, R.

Filled with Grace & Co, ), 0.4! J　Buffer A containing NaCl C25mM HEPESC pH 7,5), 20% (w#) glycerol, 0. Equilibrate the column using 75% CHAPS, 1 ml J EDTAI.

Load the above solubilized reductase preparation onto a Blue A column.

Wash the column with several column volumes of 0.4 M NaCl-containing buffer, then Further wash with 0.5M NaCl-containing buffer 8. 90% of reductase activity More than 85% of other proteins bind to the column, while more than 85% of other proteins pass through the column. 1 ．． Elute the reductase activity from the column using OM NaCl-containing buffer A. Melt. Fractions were collected and assayed for reductase activity as described in Example 1. I do it. Both aliquots containing reductase activity are pooled and stored at -70°C. typical Specifically, 30-50% of the loaded reductase activity is 1. OM NaCl buffer It is recovered by elution at .

D. Size exclusion chromatography The active fractions pooled from the Blue A column were transferred to a YM30 membrane (AmiconDivi). For ultrafiltration in a pressurized cell equipped with Concentrate by ~10 times. Typically in an amount of ~90rnl from the Blue A column The activity was eluted, concentrated to ~8-, and added to two Sephacrystals as follows. SephacrYl S-100 column. Column (2,5X7 5CI11) to 5100HR media (Pharmacia LKB Biotec hnology.

Piscataway, NJ) and buffer containing 0.5M NaCl. Equilibrate with liquid 8. The following protein standard: bovine serum albumin (66kD) , carbonic anhydrase (29kD), cytochrome C (12,4kD) and size assay using blue dextran (used to measure interstitial volume).

41nJ of concentrated sample! Apply aliquots to each 5100 columns, approximately 170/hour It develops at a linear flow velocity of Collect fractions over ~4 hours and Tase activity is measured as described in Example 1.

More than 60% of the loaded activity elutes at an apparent molecular weight of approximately 49 kD. Collected in one main peak. The volume of the pooled active fractions is approximately 30-35 -/column.

E, Affinity chromatography column (1.5 cm x ~ 2 cm) Valmitoyl-CoA agarose (Sigma Chemical Co,... St, Louis, MO) and buffer B (0, IM NaCl Equilibrate with buffer A) containing Top the active fractions pooled from the gel filtration column. Concentrate ~16x by ultrafiltration as described above. NaCl level in concentrated sample is lowered from 0.5M to approximately 0.1M by dilution with Buffer A. Color the diluted sample and then washed with several column volumes of buffer B. Then 15mM NA After washing the column with buffer B containing DH 10-, it is further washed with buffer B.

The reductor was prepared by flowing 15mM NADPH in buffer B through the column. elute the enzyme activity. Typically, material from one gel filtration column is The activity can be processed on an affinity column and applied to the column. More than 70% of the amount is recovered by elution with NADPH. Pool the active fractions and Analyze for ductase activity, protein concentration and polypeptide composition. Ta Protein concentration was determined by Bradford (AnalY, Biochem, (197 6) Commercially available dyes based on the dye binding method described by J.D. 72:248-254 (Bio-Rad Laboratories, Inc., Richm ond.

CA).

F, 5DS-PAGE analysis The polypeptide composition of the sample was determined by 5OS-PAGE (Laemmli, O., ( 1970) Nature (London) 227: 680-685) Analyze. For samples for electrophoresis, remove SDS and dithiothreitol from the stock solution. They are prepared by adding them to final concentrations of 2% and 30mM, respectively.

Approximately 50 μl of the sample was transferred to a 12% separation gel (NOVEX, San Diego, CA) onto the wells of an acrylamide gel. Bio-Labor Molecular weight standards were purchased from A-tories. Proteins are detected by silver staining (Blum et al., two major molecules with apparent molecular weights of about 52 kD and about 54 kD A polypeptide band is detected in the active sample from the affinity column and this The two polypeptide bands together account for >95% of the protein in this preparation. occupy The apparent size of the reductase enzyme in its native state is approximately 49 kD. (as measured by size exclusion chromatography as described above), so These bands represent a receptor rather than two different subunits of one enzyme. It appears to represent a related form of the ductase enzyme.

G, Plotting of proteins onto membranes. After S-PAGE, either nitrocellulose or PVDF, 1 mmabil on-P (Millipore, Bedford, MA) or ProB lot (Applied Biosystems: Foster C1t y, CA) by transferring these proteins to the membrane of either Isolate for amino acid sequencing. If the protein would then be enzymatically digested Nitrocellulose is preferred, and N-terminal sequencing and cyanogen bromide digestion are preferred. PVDF is useful for sequencing peptides generated from

l Plotting on nitrocellulose: Proteins on nitrocellulose When performing electrical plotting, the plotting time is 5-20% methanol. Typically in a buffer such as 25mM Tris, 192mM glycine in 1 to 5 hours. After electroplotting, 0.1% ( w/v) Stain the membrane for 2 minutes with Ponceau S and change 2-3 times. , 1% (V/V) acetic acid for 2 minutes per exchange. Then those membranes Store moist in heat-sealed plastic bags at -20°C. time allows Then, without freezing the plot, you can use a peptide for determining amino acid sequences as described below. Use immediately for digestion to make chido.

2. Plotting on PVDF, protein [mmobilon P] When electrically plotting P'/DF, the plotting time is 10% ( v/v) 12.5mM Tris in methanol, 15mM glycine, etc. It is usually about 1 to 2 hours in a buffer solution. After electrical plotting to PVDF, 50 0.1% (w/v) Cooma in % (v/v) methanol/10% (v/v) acetic acid Stain the membrane for 5 min with Sea Blue and change 2-3 times with 50% (V/V) methane. Color the membrane in ethanol/10% (V/V) acetic acid for 2 minutes per exchange.

The PVDF membrane was then air-dried for 30 minutes and then placed in a heat-sealed plastic bag. Store dry at -20°C. Plot on PVDF membrane, e.g. ProBlott. The tagged protein can be used directly to determine the N-terminal sequence of the complete protein. You can use it. Let ProBlott electroplot proteins The protocol is described below in Example 4A.

Example 4 - Determination of amino acid sequence In this example, plant proteins associated with acyl-CoA reductase activity A method for determining the quality amino acid sequence is described.

A. Cyanogen bromide cleavage of proteins and separation of peptides Promega, In c, Probe-Design Peptide of (Madison, W [) Described in the Separation System Technical Manual Perform cyanogen bromide cleavage on the protein of interest using the methodology described. mentioned above Plot the reductase protein onto a PVDF membrane as follows.

Protein bands were then excised from the plot and incubated in 70% (V/V) formic acid. Immerse in a solution of cyanogen bromide and incubate in this solution overnight at room temperature.

After this incubation, the cyanogen bromide solution is removed, pooled, and the Rea Using a cti-Vap evaporator (Pierce, Rockford, [L) Dry under continuous nitrogen flow. 70% (V/V) isopropanol, 0.2% (V /V) trifluoroacetic acid, 0.1mM lysine and 0.1mM thioglycolic acid Perform additional elution of the cyanogen bromide peptide using a peptide elution solvent such as Complete removal can be guaranteed. The elution solvent was then removed and the dried bromide silicon was removed. Add to the test tube containing the solution and dry as above. Use fresh elution solvent The elution operation may be repeated. Next, add 50μ to the dry peptide! ) for IPLc Add pure water and 5peed-Vac (Savant, Inc., Farmi Water is removed by evaporation in NGDALE, NY).

Schiger and von Jagow (Anal, Biochem, (1987) Animals 6:368-379]. Peptides are separated using the s/Tricine 5DS-PAGE system. 125 ~150 volts constant voltage for approximately 1 hour or until the tracking dye begins to flow out from the bottom of the gel. Run the gel until the gel is completely absorbed. Before migration, the gel was soaked in migration buffer (125mM Tris, Soak in 50 mM glycine, 10% (v/v) methanol for 15-30 minutes.

Transfer the gel to ProB1ott sequencing membrane (Applied Biosystems , Foster C1ty, CA) for 2 hours at a constant voltage of 50 volts. ing. Coomassie Blue [50% (V/V) methanol/10% (V /V) 0.1% in acetic acid] and 50% (V/V) methanol/1 Decolorize in 0% (V/V) acetic acid for 3 x 2 minutes. The membrane was then air-dried for 30-45 minutes. After that, dry and store at -20''C.

Peptides blotted onto ProBlott were coated with polybrene. Protein sequencing without the addition of filtered glass fiber filters can be introduced into the sequencer cartridge of the processor. slightly modified Reaction cycle BLOT-1 (provided by Applied Biosystems) Sequence the peptides using In addition, solution S3 (butyl chloride) is 1 and S2 (n-hebutane and ethyl acetate).

When sequencing samples blotted on ProBlott, always Two modifications are used.

B, Protease digestion and peptide separation blotting onto nitrocellulose. The extracted proteins undergo protease digestion to obtain peptides for sequencing. can be used. The method used is described by Aebersold et al. (PNAS (198 7) 84:6970) method. Reductase protein bands and An equal amount of blank nitrocellulose was added to the nitrocellulose membrane to serve as a control. The Ponceau S was removed by cutting it out and washing it several times with HPLC-grade pure water. child After washing, 0.5% polyvinylpyrrolidone (PVP-40°A) in 0.5% acetic acid was added. Add 1.0 ml of M. ldrich, Milwaukee, Wl. to the membrane section. and incubate the mixture for 30 minutes at 37°C. Completed PVP-40 To completely remove the nitrocellulose, wash the nitrocellulose piece with a large amount of HPLC grade water8. X5J), check the absorbance of the washing solution at 214 nm with a spectrophotometer. Also , if the band is not cut into small sections until after PVP-40 treatment and washing, PVP-40 are more easily removed. Those two changes are a hindrance regarding PVP-40. Eliminate the problem.

Then add a suitable digestion buffer, e.g. 100mM bicarbonate of tryptic digestion buffer. 25mM charcoal in thorium pH 8.2 or endoproteinase gluc buffer Suspend membrane sections in ammonium acid/1 mM EDTA, pH 7,8. Disappear Add acetonitrile to the mixture to a concentration of 5-10% (V/v). . Dilute protease in digestion buffer and typically 1:10 (w/w ) to the digestion mixture at a protease-to-protein ratio of Digested product 1 Incubate from 8:00 to 24:00. For example, tryptic digests are heated at 37°C. Incubate and endoproteinase gluc digest ink at room temperature. cuveate. Similarly, other proteinases such as 1ysc and aspN Reductase may be used to digest proteins. Individual digestion buffer conditions vary protocols for digestion, peptide isolation, purification and sequencing. The method is substantially similar to that described for digestion with trypsin and gluc. It's the same.

After overnight incubation, lOμβ of 10% (v/v) trifluoroacetic acid (TFA) or 1μ! The digestion reaction is stopped by the addition of +00% TFA.

Remove the digestion mixture from the nitrocellulose piece and cut the nitrocellulose piece into a 100μ Wash the β digestion buffer 1-5 times with 15-10% acetonitrile and apply them to 5 pe Concentrate to a volume of less than 100 μl in an ed-Vac. Applied Bios systems (Foster C1ty, CA) 130 type high performance liquid chrome Vydac reverse phase C18 column (2, Separate the peptides on a 1 am x 100 mm). Elute the peptide The mobile phase used for is as follows: Buffer A: 0.1mM sodium phosphate, p H2,2+ Buffer B 0% Acetonitrile 10.1mM Sodium Phosphate, p H2,2° 10-55% buffer B over 2 hours, 55-75% buffer over 5 minutes A 3-step gradient of solution B and 75% buffer B constant for 15 minutes at a flow rate of 50 μl/min. Use it quickly. Peptides were detected at 214 nm, collected manually, and then ``Save as C.

N-terminal sequence analysis of C0 proteins and peptides All sequence analyzes were performed using Appli ed Biosystems 477A Pu1sed-LiquidPhas Performed by Edman decomposition on the eProtein 5equencer. Applicable The phenylthiohydantoin (PTH) amino acid produced by the sequencer is Online Applied Biosystems 120A PTHAna Analyze by lyzer. Collect data and use Apple MaCintO3h Applied Biosys-tems 61OA type data analysis system for , and also PE NELSON.

ACCESS” CHROM from Inc. (Cuperfino, CA) Integrate on Digital Microvax using software. array data Record the data from the chart recorder and input it to the PTH analyzer. Accept and verify using quantitative data obtained from Model 610A software. Ru.

All sequence data were analyzed by two operators with the help of a data analysis system. Read independently.

For peptide samples obtained as HPLC peak-off, sequence Polybrene-coated fiberglass fibres, subjected to three pre-cycles in a Luther (Applled Biosystems, Foster C1ty.

CA) Place the sample on top. For peptides that have been reduced or alkylated, A portion of the PTH-amino acid product from the sequencer cycle was transferred to liquid scintillator. count in the action counter.

For protein samples electroblotted onto Immob i Ion-P cut out the band of interest and then pre-run it through a pre-cycle as described above. onto a polybrene-coated fiberglass filter, and according to the manufacturer's instructions. Assemble the reaction cartridge according to the instructions.

For protein samples electroblotted onto ProBIotto, No glass fiber filter is required.

To obtain protein sequences from small samples (5-30 pmol), Temp st and Riviere (Anal, Biochem, (1989) 183: 477A conversion cycle and 12OA analyzer described by 2903 Use riser.

D, Amino acid sequence of reductase peptide Purified reductase preparation in 5D Apply S-PAGE to separate 54 kD and 56 kD proteins. separated The material was transferred to a nitrocellulose-type membrane ([mmobilon N)] and Po Stain with nceau Red to locate the band. 56 kD or 54 Trypsinize the cut section of the plot containing either of the kD proteins. , tryptic peptides are separated by reverse phase HPLC. Each reductase protein Sequence information obtained from several peptides from the quality is given in Table 2 below.

Table 2: Peptide sequences of 54 kD and 56 kD reductase proteins 56 kD reductase peptide 56 kD reductase peptide 1) A Engineering LVTGATGSLAK l) AILVTGATGSLAK2) LQNE xFGKELFK 2) LGLD engineering NVEK3) VTVVPGDITGED L 3) T from NVPVYYG4) LGLD Engineering NVEK 4) Yl /EP VTYxVGSSAAN5) TIDMnVYYGK 5) LVDr YKp6) YVEP VTYHVGS SAANPM 6) EG engineering V EAfl”Yr sword LSALPEMAJ (R7) AINhaDYFL8) LVD work YK 8) THFPGVt/E3 (VL9) EG work VEADl’F MFD 10) A engineering NWED YFLKTxFPGVVE xVL two reductors zeta Protein homology is evident from the above peptide sequences. 54 kD protein All sequenced peptides from are also found in the sequenced 56 kD peptide . The determined amino acid sequence and cDNA encoding 56 kD reductase are There is one discrepancy between the deduced reductase amino acid sequence (I]) be. Amino acid 460 is serine according to the cDNA sequence data. 54 and The respective information from 56 kD peptides 6 and 9 indicates that this position is a glycine. Show that something is true.

Southern block of jojoba genomic DNA digested with four different restriction enzymes Losotho analysis uses one main vector that hybridizes to the reductase cDNA probe. band and one subband.

Example 5 - Jojoba cDNA library Jojoba collected 80 to 90 days after flowering Construction of a jojoba embryo cDNA library from poly(A) RNA isolated from jojoba embryos The preparation is described.

Isolate RNA. In this method, all steps are performed at 4°C unless otherwise specified. . ] The Og tissue was placed in a Waring Blengu under liquid nitrogen until the tissue became a fine powder. Grind with. After evaporating the liquid nitrogen, add 170-ml extraction buffer (200mM Tris pH 9, 0, 160mM KCl, 25mM EGTA, 701 11M MgCl□, 1% Triton X-100, 0.5% deoxycor Sodium Runate.

1mM spermidine, 10mM β-mercaptoethanol and 500mM sucrose) and homogenize the tissue for approximately 2 minutes. Transfer the homogenate to sterile gauze Filter through and centrifuge at 12,000×g for 20 minutes. Sterilize the upper groove with 500- Decant into a flask and add 1/19 volume of 20% surfactant solution ( 20%Br1j 35.20%Tween 40.20%No1detP-40 w/v) at room temperature. The solution was stirred at medium speed for 30 min at 4 °C, The supernatant is then centrifuged at J2.000Xg for 30 minutes.

Approximately 30-aliquots of the supernatant were aliquoted into sterile T160 centrifuge tubes and diluted with 40mM Tris. pH 9, 0, 5mM EGTA, 200mM KCl, 30n+M Mg C1z, solution 7 containing 1.8M sucrose and 5mM β-mercaptoethanol Overlay with rnl. Fill the test tube to the top with extraction buffer and place it in a Ti60 rotor. Centrifuge at 60,000 rpm for 4 hours at 4°C in a motor. After centrifugation, top Aspirate the groove and add 0.5- resuspension buffer (40mM Tris pH 9,0, 5mM EGTA, 200mM KCI.

Add 30mM MgCl□, 5mM β-mercaptoethanol) to each test tube. do. Place the tube on ice for 10 minutes, then thoroughly resuspend the pellet and remove the file. It is then centrifuged at 120Xg for 10 minutes to remove insoluble material. 20m M Tris pH 7, 6, 200mM EDTA, 2% N-Lauryl Sarcosi Add 1 volume of solution of autolyzed 1/i proteinase in nate to the supernatant. , incubate the mixture for 30 minutes at room temperature.

RNA was extracted by adding 1/10 volume of sodium acetate and 2 volumes of ethanol. Let it settle. After several hours at -20°C, 12.000Xg for 30 minutes at 4°C. Pellet the RNA by centrifugation. Transfer this pellet to 10ml of TE Resuspend in buffer solution (10mM Tris, 1ml 1J EDTA) and add the same volume of T Extract with ris pH 7.5 saturated phenol. io, oooxg at 4°C The phases were separated by centrifugation for 20 minutes, the aqueous phase was removed, and the organic phase was added to 1 volume of TE buffer. Re-extract with liquid. The aqueous phases are then pooled and extracted with 1 volume of chloroform. centrifugal The aqueous phase was precipitated with ethanol as described above, and the polyliposomes were separated. Obtain muRNA.

The RNA was added to a high temperature buffer (0.5M NaCl, 20mM Tris pH 7.5 , 1mM EDTA.

Passed through a cellulose column (Sigma-cell 50) in 0.1% 5DS). This removes polysaccharide contaminants during liposomal RNA preparation. The contaminants are ram and the RNA is collected in the eluate. Pool the eluate fractions and RNA is ethanol precipitated. Resuspend the precipitated total RNA in a small volume and add the oligo Polyadenylated RNA is isolated by application to a d(T)-cellulose column.

B. Construction of cDNA library in plasmid vector Polyadenylated RN cDNA in plasmid cloning vector pcGNI703 using A. Create a library. Plasmid vector 1) CGNI703 is a commercially available Cloning vector Bluescribe M13-(Stratagene Derived from Cloning System; San Diego, CA) and is produced as follows. Digestion with BamHl, mung bean endnu Bluescribe M13 by treatment with crease and blunt end ligation -Changed the polylinker to create BamHI deletion plasmid pcGN1700 do. Digest pcGN1700 with EcoRI (5stl adjacent restriction sites), and BamHl, Pstl, Xba[, Apa[ and SmaI restriction section synthetic phosphorus with a 5′ overhang of AATT and a 3′ overhang of TCGA. Anneal with the car. Insertion of the linker into pcGN+700 The Rr site was deleted and the 5stl (Lbashiba “S”) found in Bluescribe was ac [also referred to as “)” and the new linker contained on the linker Introducing restriction sites. The resulting plasmid pcGN1702 was digested with Hindu. , and make blunt ends with Flenow enzyme. Linear DNA was partially digested with PvuI [T 4. Ligate in a dilute solution using DNA ligase. lac promoter region Select a transformant in which the region has been deleted. 1) CGN1703) and add it. Use as a mid cloning vector.

Briefly, the cloning method for cDNA synthesis is as follows. plastic The Sumido cloning vector was digested with Sst[, and the resulting 3' overhanging sticky end was homopolymers using terminal deoxynucleotidyl transferase to Attach a T-tail. The tailed plasmid was transformed into an oligo(dA)-cellulose Undigested plasmid or tailless plasmid by chromatography Separate from. The resulting vector is covalently linked to one end of the vector plasmid. Serves as a primer for the synthesis of a synthetically attached cDNA second strand. cDN A-mRNA-vector complex is tertiary in the presence of deoxyguanosine triphosphate. G-tails are attached to the ends of cDNAH by treatment with terminal transferase.

BamH [Extra cDNA adjacent to the site - [11RNA complex removed by digestion, with a BamHr sticky end on one end and a G-tail on the other end. The cDNA-mRNA-vector complex containing the remaining cDNA-mRNA-vector complex is left behind. This complex is 5' BamH [sticky end, restriction enzyme Notl, EcoR [and 5stl recognition site] cyclization using an annealed synthetic cyclization linker with a sequence and a 3' C-tail. urge After ligation and repair, the circular complex was used to transform E. coli strain DH5a (BRL, Gaithersburg, MD) was transformed into a cDNA library. -Create a Jojoba [EcDNA Bank has an average cDNA insert of approximately 500 base pairs. Approximately 1.5 NA The cloning vector λZAP (1/EcoRr (Stratag Generate a cDNA library at EN, San Diego, CA) do. The library was prepared according to the protocol provided by the manufacturer, D? i'A and bacterial strains. Gigapack as well according to the manufacturer's instructions Clone using Gold packaging extract (Stratagene) Packaging.

The cDNA library prepared using this method has an average cDNA insert of approximately 400 base pairs. Contains approximately IX10' clones with the same fragment size.

D. Isolation of reductase cDNA Using PCR technology using primers designed from the reductase peptide sequence , screening a jojoba library in the pcGNI703 bacterial vector An approximately 1 kb portion of the reductase nucleic acid sequence is generated.

Techniques known in the art, such as those described by Maniatis et al., supra. The library is screened using the technique. 56 kD reductor zeta The protein clone pcGN7571 was obtained and its DNA sequence was determined. Ru. The nucleic acid and amino acid sequences of pcGN7571 are provided in FIG.

Reductor in plant cells using the 5' and 3' e-regions from the napin gene A construct for expressing the enzyme is prepared as follows.

Nuvin expression cassette pCGN180g is co-pending U.S. Patent Application No. 071550. No. 804, this publication is incorporated herein by reference. . 9CGN1808 by transferring only the expression sequence and transferring the antibiotic resistance marker. be modified to include flanking restriction sites to ensure stability, and the binary vector, e.g. pcGN1557 (McBride and Summerfelt.

above). Kpnl, Not[, and Hind[II restriction site-containing compounds. The oligonucleotides are annealed and only one Hind[I[ site is Unique Hindll of pcGN1808 to be played conclude. The resulting plasmid pcGN3200 is confirmed by sequence analysis. and unique Hind [Il, Not [ and Kpn [Contains restriction sites.

1) Digest CGN3200 with Hindl[[ and 5acl; and pIc19R digested with Sac (Marsh et al. (1984) Gene 32:481-485) to increase the size of the Nuvin expression cassette. The portion is subcloned to create pcGN3212. nuvin promoter I) CGN3200, Sac [site and and two primers flanking the junction of the napin 5' promoter, and the pc by PCR using the pUC backbone of PCGN3200 from the GN1808 construct. Reconfigure. The forward primers were C1an, Hindl[1, Notl and Kp nl restriction site and nucleotides 408-423 of the nubbin 5' sequence (snl coR V site) and the reverse primer contains the unique Sa c[comprising the complement to the napin sequence 718-739 containing the site. PCR is manufactured by Using a Perkin E1mer/Cetus thermocycler according to manufacturer's instructions went. The PCR fragment was converted into a blunt fragment and pUc8 (V ieira and Messlngru. pcGN3217 over the Nubin insert Sequencing showed that no incorrect nucleotides were introduced by PCR. to confirm.

pcGN3217 was digested with C1aI and 5acl and quenched with C1al and Sac[ I) By ligating with converted pCGN3212, The Napin 5' sequence is linked to the remainder of the Napin expression cassette. Obtained expression skein Digest the cut pcGN3221 with HindlI and gel the napin expression sequence. purified and ligated with old ndlli digested p[c20H (Marsh, supra). Close. The final expression cassette was placed in pcGN1808 in an ampicillin resistant background. 1.725 nubbins 5' and 1.2653' adjustments essentially identical to those found in pCGN3223 containing the sequence. The regulatory region is Hindll[, Notl and Kpn1 restriction sites, and are flanked by 5' non-hood regions and 3 'Unique 5ai1. between non-coding regions. Bgl[1, Pst[ and Xh o [The cloning site is located.

Reductase cDNA I) CGN7571 was converted to Sph[(base +594-1 599 in the 3' untranslated sequence) and injected Sal [Insert linker. The resulting plasmid was digested with BamH[ and 5alI, The fragment containing the ductase cDNA was gel purified and the Bg1[r/Xho Cloned into pCGN3223 (Navin expression cassette described above) digested with to obtain pCGN7585.

Hin of pCGN7585 containing the nuvin 5'/reductase/nuvin 3' construct. The dl[I fragment was digested with Hindr[[pcGN1578 (McBri de and Summerfelt, supra) for plant transformation. Obtain the binary vector I) CGN7586. Holsters et al. Mat, Gen, Genet.

B. Plant transformation Seeds high in erucic acid, such as cultivars Reston, and Seifolia Seeds of canola hard variety of rapeseed (Brassica napus) with 95% Soaked in tanol for 2 minutes, 1.0% sodium hypochlorite with -drops of Tween 20 Surface sterilize for approximately 30 minutes in a sterile distilled water solution and rinse three times in sterile distilled water. Next Pyridoxine (50 μg/A’), nicotinic acid (50 μg/β), glycidi ( 20011g7G and 0.6% Phytagar (Gibco) pH 5, Murashige minimal organic medium (Gibco; Gran Seed in Magenta boxes with d[5land, NY]. 22 Approximately 45-55 microns/square meter/m2 in a cell culture room at ~24°C. In a 16-hour photoperiod using cold fluorescent red light at an intensity of to germinate the seeds.

Excise hypocotyls from 6- to 7-day-old seedlings, cut into approximately 2- to 4-length sections, and Boar feeder plate CHharsh et al., 5science (1985) Thor, 1.3 ■/l Thiamine-HCl, 200 ■Nio PO4, 3% solution Sugar, 2.4-D C1,0mg/I), 0.6% (W/V) Phytaga A medium (MSO) containing R and adjusted to pH 5.8 before autoclaving /I10 medium) in a Petri dish (100 x 25 mm) containing 1,0- Prepare the day before use by inoculating a cloudy culture. IO- culture +00 WLl fresh MS medium [as described for feeder plates, but 2.4-D (0,2■/β), containing kinetin (0,1■/l)] Tobacco suspension cultures are subcultured weekly. In experiments without supporting cells, Hypocotyl explants are cut and placed on filter paper disks on top of MSO/I10 medium. all Hypocotyl explants were subjected to a series of intensities of 30μEm-”S-’ to 65μEm-”S-’. Pre-incubate on a feeder plate for 24 hours at 22-24°C under continuous light. Bate.

A. tumefaciens (A. tumefaciens) containing a binary plasmid ns) A single colony of εHA 101 strain was transferred to 5-MG Rubros, where 3 Grow overnight at 0°C. Hypocotyl explants were diluted to lXl0” bacteria/− Soak in about 1 liter of MG Lebros containing and feeder plate after about 10 minutes. Place it on top. MG Lebros contains 5g of mannitol and 1g of L-glue per If. Tamic acid or 1.15g monosodium glutamate, 0.25g KHzPO 4,0,10g of NaCl,0,10g of Mg5 (L・7H20,1■ biotin 5 g of tryptone and 2.5 g of yeast extract, the broth has a pH of 7, Adjusted to 0. After 48 hours of co-incubation with Agrobacterium , hypocotyl explants were treated with filter-sterilized carbenicillin (500 μ/I!, autoclave). (added after sterilization) and kanamycin sulfate (25■/I!, Boeh ringer Mannheim: [ndianapolis, IN) B50/I10 callus induction medium containing (Img/A' 2.4-D, 0.6% Ph ytagar supplemented with B5 salts and vitamins).

After culturing for 3 to 7 days under continuous light of approximately 85μEm”S-’, callus tissue was observed on the cut surface. Once recognized, the hypocotyl explants were placed in shoot induction medium B5BZ (3/I! benzyl chloride). Supplemented with minoprine, 1 ■/l zeatin, 1% sucrose, 0.6% Phytagar. B5 salts and vitamins added and pH adjusted to 5.8). This medium also contains carbenicillin (500■/j7) and kanamycin sulfate (25■/1) . Subculture hypocotyl explants into fresh shoot induction medium every two weeks.

Shoots regenerate from the hypocotyl callus after 1 to 3 months. Small (green with the height of the fan) Cut the shoots from the callus and add B5 salts and vitamins, 1% sucrose, carbenicili. Kanamycin sulfate (50 mg/I and 0.6% (w/1)) v) Place on medium containing Phytagar (after 2-4 weeks, shoots remain green) cut out from the root, and add rooting induction medium (85 salts and vitamins, 1% sucrose). , 2■/i indolebutyric acid, 50■/l kanamycin sulfate and 0.6% P. Transfer to the magenta box containing hytagar.

The above results obtained by Agrobacterium-mediated transformation as described by , a solubilized seed plant fatty acyl that is active in the formation of fatty alcohols. We demonstrate that the ductase protein can be obtained. acyl reductase Methods of obtaining the protein and its amino acid sequence are provided. In addition, the reductor Also provided are enzyme nucleic acid sequences. By manipulating those nucleic acid sequences, may provide for transcription of the sequence and/or expression of the reductase protein.

Reductase proteins can be used in a variety of applications.

All publications and patent applications cited herein may be cited as individual publications or patent applications. It is pointed out that the patent application is expressly and individually incorporated herein by reference. is incorporated herein by reference as if by reference.

The present invention has now been described in some detail and by examples for purposes of clarity and understanding. However, in light of the technology of the present invention, the spirit and scope of the appended claims It will be apparent to those skilled in the art that certain changes and improvements may be made without departing from the scope. It should be easily obvious.

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C< <Φ's place Qo o:l E-1■l! −＋−〇〇　ToΦ　Ql、IF-1 Φ Tofuchi (H 0- U mountain E@-To~ 0 Q- C1) GIQ, O≦菰 <8 VV a＜ma ni-riichitoto (, -1(J-hioJl-+　u　O(jυ←市　く＞　hee　〇-itoΦ o>hhi<H<E@desire Σ ■Ri Q’s Qα Q Lot α’s <>< of <ri> ψ ＜, -z　＜,,5　C:l＜　＜＜　a＜㽜(ri:110　←≧　I+!knee Q−E−田←ωOC), −I　E”zu　←Φ　○H)−＋＋Jw　CDCD　( j＞　＜Σ　Qkuto0　0ののくり　Qα　Poor! < of 0″ Hungry? Knee 2 (ri < CD Qoyama Summary book fatty acyl reductase According to the invention, species that are partially purified and active in fatty acyl substrates Seedling fatty acyl reductase proteins are provided. Of particular note are the approximately 5 Jojoba embryo reductasetan has molecules of 4 and 52 kD. the Amino acids and nucleic acids that can be obtained from reductases such as .

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Claims (30)

[Claims] 1. Partially purified seed plant fatty acyl reductase comprising a fatty acyl substrate Said reductase is active in the formation of fatty alcohols from. 2. 2. The reductase of claim 1, which has activity against fatty acyl-CoA substrates. 3. Fatty acids having a carbon chain of the formula C2x (where X is selected from 8 to 12) 2. The reductase of claim 1, which has activity against a single substrate. 4. 2. The reductase of claim 1, wherein said reductase is NADPH dependent. 5. 2. The reductase of claim 1, which is obtainable from jojoba embryos. 6. 5. Consisting essentially of said reductase having a molecular weight of about 55 kD. reductase. 7. A recombinant construct, comprising: a recombinant composition for forming fatty alcohols from fatty acyl substrates; a nucleic acid sequence encoding at least a portion of a fatty acyl reductase that is active in A recombinant construct comprising. 8. 7. The reductase has activity against fatty acyl-CoA substrates. recombinant constructs. 9. The reductase is a carbon of the formula C2x, where X is selected from 8 to 12. 8. The recombinant composition of claim 7, which has activity towards fatty acyl substrates having chains. 10. 8. The recombinant construct of claim 7, wherein said reductase is NADPH dependent. 11. The recombinant composition of claim 7, wherein the nucleic acid sequence is obtainable from a seed plant. . 12. 8. The recombinant composition of claim 7, wherein said nucleic acid sequence is obtainable from jojoba. 13. 5 providing for at least transcription of said reductase coding sequence in a host cell. 8. The recombinant construct of claim 7, further comprising non-coding sequences. 14. a 5' non-coding sequence that provides for expression of the reductase coding sequence in the host cell. 8. The recombinant construct of claim 7 further comprising the sequence. 15. The reductase coding sequence is antisense to the 5' non-coding sequence. 14. The recombinant construct of claim 13, wherein the recombinant construct is in the direction of 16. Any of claims 13, 14 or 15, wherein the host cell is a plant cell. One item of wisdom change composition. 17. A cell comprising a recombinant construct according to claim 7. 18. A plant cell comprising a recombinant construct according to claim 7. 19. Brassicaceae (Brassicaceae) comprising the recombinant construct according to claim 7. a) Plant cells. 20. A prokaryotic cell comprising a seed plant fatty acyl-CoA reductase. 21. A method for producing fatty acyl reductase in a host cell, the method comprising: Fatty acyl Reda that is active in the formation of fatty alcohols from fatty acyl substrates comprising a nucleic acid sequence encoding a reductase and said reductase encoding sequence. a host cell containing the recombinant construct under the control of functional regulatory elements in said cell. , growing under conditions that will cause expression of said reductase sequence. A method comprising. 22. 22. The method of claim 21, wherein the host cell is a seed plant embryo cell. 23. 22. The method of claim 21, wherein the host cell is of the family Brassica. Law. 24. 22. The method of claim 21, wherein the nucleic acid sequence is obtainable from a seed plant. Method. 25. a solubilized seed plant fatty acyl reductase comprising a fatty acyl substrate; Said reductase is active in the formation of fatty alcohols from. 26. 26. The reductase of claim 25, which has activity against fatty acyl-CoA substrates. . 27. fatty acids having a carbon chain of formula C2x, where X is selected from 8 to 12; 26. The reductase of claim 25, which has activity against sil substrates. 28. 26. The reductase of claim 25, wherein said reductase is NADPH dependent. . 29. 26. The reductase of claim 25, obtainable from jojoba embryos. 30. Claim consisting essentially of said reductase having a molecular weight of about 55 kD. 29 reductase.