Protein compositions having isoprene polymerization activity and uses thereof

Abstract

The present invention provides a means for efficiently producing natural rubber and achieving stable securing, with the aim of securing rubber resources. The present invention provides a protein composition comprising (A-1) a protein having the same activity as CPT6 or (A-2) a protein having the same activity as CPT7 and comprising (B) a protein having the same activity as CPTL, a lipid membrane construct comprising the above composition, a method for producing the same, a cell expressing the proteins constituting the above composition, a method for producing the same, and a method for producing an isoprene polymer compound using any one of these.

Description

Technical Field

[0001] This invention relates to protein compositions having isoprene polymerization activity and their uses, and more specifically, to protein compositions having isoprene polymerization activity, lipid membrane constructs and cells comprising the same, and methods for manufacturing isoprene polymeric compounds using the same. Background Technology

[0002] Natural rubber, primarily composed of cis-polyisoprene found in the sap of rubber trees, is widely used in various sectors including the automotive and construction industries, and global demand is expanding. However, because natural rubber is harvested from rubber trees, it is susceptible to supply instability and quality variations, potentially posing significant business risks. Therefore, efforts are underway to develop technologies for the artificial production of natural rubber.

[0003] Patent Document 1 and Non-Patent Document 1 describe a method for manufacturing polyisoprene that includes a binding step of binding rubber particles with the gene expression products of specified proteins (HRT1, HRTBP, and REF) related to the biosynthesis of natural rubber in vitro.

[0004] Existing technical documents

[0005] Patent documents

[0006] Patent Document 1: Patent No. 6586693

[0007] Non-patent literature

[0008] Non-patent literature 1: Yamashita s. et al. eLife 2016;5:e19022. DOI: 10.7554 / eLife.19022 Summary of the Invention

[0009] The problem that the invention aims to solve

[0010] The techniques described in Patent Document 1 and Non-Patent Document 1 cannot adequately produce polyisoprene and cannot be fully practically applied. Furthermore, such techniques present the following problems: when rubber particles need to be collected from plants, the collection and processing of the rubber particles is complex; and because they are extracts from plants, stabilizing the quality of the rubber particles is difficult.

[0011] This invention provides an efficient means of producing natural rubber and ensuring its stability, with the aim of ensuring stable rubber resources.

[0012] Methods for solving problems

[0013] According to the present invention, the following [1] to

[24] are provided.

[0014] [1] A protein composition having isoprene polymerization activity, wherein the protein composition comprises:

[0015] (A-1) One or more proteins selected from proteins containing the amino acid sequence of sequence number 6 and proteins containing an amino acid sequence that has more than 90% identity with the amino acid sequence of sequence number 6 and has the same activity as CPT6, or

[0016] (A-2) One or more proteins selected from proteins containing the amino acid sequence of sequence number 8 and proteins containing an amino acid sequence that has more than 90% identity with the amino acid sequence of sequence number 8 and has the same activity as CPT7.

[0017] Furthermore, the protein composition comprises:

[0018] (B) One or more proteins selected from proteins containing the amino acid sequence of sequence number 10 and proteins containing an amino acid sequence that is more than 90% identical to the amino acid sequence of sequence number 10 and having the same activity as CPTL.

[0019] [2] The composition according to [1] satisfies at least one of the following:

[0020] (A-1) is a protein encoded by a polynucleotide (a-1), wherein the polynucleotide (a-1) is selected from one or more polynucleotides containing the base sequence of sequence number 5 and containing a base sequence having more than 90% identity with the base sequence of sequence number 5 and encoding a protein having the same activity as CPT6.

[0021] (A-2) is a protein encoded by a polynucleotide (a-2), wherein the polynucleotide (a-2) is selected from one or more polynucleotides containing the base sequence of sequence number 7 and containing a base sequence having more than 90% identity with the base sequence of sequence number 7 and encoding a protein having the same activity as CPT7.

[0022] (B) is a protein encoded by a polynucleotide (b), which is selected from one or more polynucleotides including the polynucleotide containing the base sequence of sequence number 9 and the polynucleotide encoding the protein described below, which is encoded by a polynucleotide containing a base sequence having more than 90% identity with the base sequence of sequence number 9 and has the same activity as CPTL.

[0023] [3] The composition according to [1] or [2] further comprises CPT family proteins other than CPT6, 7 and CPTL, REF family proteins and SRPP family proteins.

[0024] [4] The composition according to [1] further comprises at least one of the following:

[0025] (C) One or more proteins selected from proteins containing the amino acid sequence of sequence number 2 and proteins containing an amino acid sequence that has more than 90% identity with the amino acid sequence of sequence number 2 and has the same activity as CPT1.

[0026] (D) One or more proteins selected from proteins containing the amino acid sequence of sequence number 4 and proteins containing an amino acid sequence that has more than 90% identity with the amino acid sequence of sequence number 4 and has the same activity as CPT2.

[0027] (E) One or more proteins selected from proteins containing the amino acid sequence of sequence number 12 and proteins containing an amino acid sequence that has more than 90% identity with the amino acid sequence of sequence number 12 and has the same activity as REF1.

[0028] (F) One or more proteins selected from proteins containing the amino acid sequence of sequence number 14 and proteins containing an amino acid sequence that has more than 90% identity with the amino acid sequence of sequence number 14 and has the same activity as REF2.

[0029] (G) One or more proteins selected from proteins containing the amino acid sequence of sequence number 16 and proteins containing an amino acid sequence having more than 90% identity with the amino acid sequence of sequence number 16 and having the same activity as REF8, and

[0030] (H) One or more proteins selected from proteins containing the amino acid sequence of sequence number 18 and proteins containing an amino acid sequence that is more than 90% identical to the amino acid sequence of sequence number 18 and having the same activity as SRPP1.

[0031] [5] The composition according to [4] satisfies at least one of the following:

[0032] (C) is a protein encoded by polynucleotide (c), which is selected from one or more polynucleotides selected from polynucleotides containing the base sequence of sequence number 1 and polynucleotides encoding the following protein, which is encoded by a polynucleotide containing a base sequence having more than 90% identity with the base sequence of sequence number 1 and having the same activity as CPT1.

[0033] (D) is a protein encoded by polynucleotide (d), which is selected from one or more polynucleotides selected from polynucleotides containing the base sequence of sequence number 3 and polynucleotides encoding the following protein, which is encoded by a polynucleotide containing a base sequence having more than 90% identity with the base sequence of sequence number 3 and having the same activity as CPT2.

[0034] (E) is a protein encoded by polynucleotide (e), which is selected from one or more polynucleotides selected from polynucleotides containing the base sequence of sequence number 11 and polynucleotides encoding the following protein, which is encoded by a polynucleotide containing a base sequence having more than 90% identity with the base sequence of sequence number 11 and having the same activity as REF1.

[0035] (F) is a protein encoded by polynucleotide (f), which is selected from one or more polynucleotides selected from polynucleotides containing the base sequence of sequence number 13 and polynucleotides encoding the following protein, which is encoded by a polynucleotide containing a base sequence having more than 90% identity with the base sequence of sequence number 13 and having the same activity as REF2.

[0036] (G) is a protein encoded by a polynucleotide (g), said polynucleotide (g) being selected from one or more polynucleotides selected from the polynucleotides containing the base sequence of sequence number 15 and the polynucleotides encoding the following protein, said protein being encoded by a polynucleotide containing a base sequence having more than 90% identity with the base sequence of sequence number 15 and having the same activity as REF8, and

[0037] (H) is a protein encoded by a polynucleotide (h), which is selected from one or more polynucleotides including the polynucleotide containing the base sequence of sequence number 17 and the polynucleotide encoding the protein described below, which is encoded by a polynucleotide containing a base sequence having more than 90% identity with the base sequence of sequence number 17 and has the same activity as SRPP1.

[0038] [6] The composition according to any one of [1] to [5], wherein,

[0039] At least one of the proteins (A) to (H) is a protein derived from the rubber tree.

[0040] [7] A lipid membrane construct comprising the composition described in any one of [1] to [6] and phospholipids.

[0041] [8] The construct described in [7] is a lipid bilayer membrane construct.

[0042] [9] The construct described in [7] or [8] is a lipoprotein body.

[0043]

[10] A method for manufacturing a lipid membrane construct, wherein,

[0044] The lipid membrane construct is any one of [7] to [9],

[0045] The method includes a step of expressing a polynucleotide encoding a protein constituting any one of the compositions described in [1] to [6] in a cell-free protein production system in the presence of a phospholipid-containing raw material.

[0046]

[11] According to the method for manufacturing the lipid membrane construct described in

[10] , wherein,

[0047] The polynucleotide encoding the protein of any one of the compositions described in [1] to [6] comprises:

[0048] (a-1) One or more polynucleotides selected from the polynucleotide sequence containing sequence number 5 and polynucleotides containing a sequence having more than 90% identity with the sequence number 5 and encoding a protein having the same activity as CPT6, or

[0049] (a-2) One or more polynucleotides selected from the polynucleotides containing the base sequence of sequence number 7 and polynucleotides containing a base sequence having more than 90% identity with the base sequence of sequence number 7 and encoding a protein having the same activity as CPT7, and

[0050] (b) One or more polynucleotides selected from the polynucleotides containing the base sequence of sequence number 9 and the polynucleotides encoding the following proteins, said proteins being encoded by polynucleotides containing a base sequence having more than 90% identity with the base sequence of sequence number 9 and having the same activity as CPTL.

[0051]

[12] The method for manufacturing the lipid membrane construct according to

[10] or

[11] , wherein,

[0052] The polynucleotide is a polynucleotide encoding a protein constituting the composition described in [3] or [4], further comprising:

[0053] (c) A polynucleotide selected from the polynucleotide containing the base sequence of Serial Number 1 and one or more polynucleotides encoding the following protein, wherein the protein is encoded by a polynucleotide containing a base sequence having more than 90% identity with the base sequence of Serial Number 1 and having the same activity as CPT1.

[0054] (d) A polynucleotide selected from the polynucleotide containing the base sequence of Serial Number 3 and one or more polynucleotides encoding the following protein, wherein the protein is encoded by a polynucleotide containing a base sequence having more than 90% identity with the base sequence of Serial Number 3 and having the same activity as CPT2.

[0055] (e) One or more polynucleotides selected from the polynucleotide sequence containing sequence number 11 and the polynucleotide encoding the protein described below, wherein the protein is encoded by a polynucleotide containing a base sequence having more than 90% identity with the base sequence of sequence number 11 and having the same activity as REF1.

[0056] (f) One or more polynucleotides selected from the polynucleotide sequence containing sequence number 13 and the polynucleotide encoding the protein described below, wherein the protein is encoded by a polynucleotide containing a base sequence having more than 90% identity with the base sequence of sequence number 13 and having the same activity as REF2.

[0057] (g) One or more polynucleotides selected from the polynucleotide sequence containing sequence number 15 and the polynucleotide encoding the protein described below, wherein the protein is encoded by a polynucleotide containing a base sequence having more than 90% identity with the base sequence of sequence number 15 and having the same activity as REF8, and

[0058] (h) One or more polynucleotides selected from the polynucleotide containing the base sequence of sequence number 17 and the polynucleotide encoding the protein described below, which is encoded by a polynucleotide containing a base sequence having more than 90% identity with the base sequence of sequence number 17 and having the same activity as SRPP1.

[0059]

[13] The method for manufacturing a lipid membrane construct according to any one of

[10] to

[12] , wherein the raw material containing phospholipids contains liposomes and the lipid membrane construct is a lipoprotein body.

[0060]

[14] A cell that expresses proteins constituting any one of the compositions described in [1] to [5].

[0061]

[15] The cell according to

[14] comprises an expression unit that contains a polynucleotide encoding a protein contained in any of the protein compositions described in [1] to [6].

[0062]

[16] The cell according to

[14] or

[15] , wherein the polynucleotide comprises:

[0063] (a-1) One or more polynucleotides selected from the polynucleotide sequence containing sequence number 5 and polynucleotides containing a sequence having more than 90% identity with the sequence number 5 and encoding a protein having the same activity as CPT6, or

[0064] (a-2) One or more polynucleotides selected from the polynucleotides containing the base sequence of sequence number 7 and polynucleotides containing a base sequence having more than 90% identity with the base sequence of sequence number 7 and encoding a protein having the same activity as CPT7, and

[0065] (b) One or more polynucleotides selected from the polynucleotides containing the base sequence of sequence number 9 and the polynucleotides encoding the following proteins, said proteins being encoded by polynucleotides containing a base sequence having more than 90% identity with the base sequence of sequence number 9 and having the same activity as CPTL.

[0066]

[17] The cell according to any one of

[14] to

[16] , wherein the polynucleotide is a polynucleotide encoding a protein constituting the composition of [3] or [4], further comprising at least one of the following:

[0067] (c) A polynucleotide selected from the polynucleotide containing the base sequence of Serial Number 1 and one or more polynucleotides encoding the following protein, wherein the protein is encoded by a polynucleotide containing a base sequence having more than 90% identity with the base sequence of Serial Number 1 and having the same activity as CPT1.

[0068] (d) A polynucleotide selected from the polynucleotide containing the base sequence of Serial Number 3 and one or more polynucleotides encoding the following protein, wherein the protein is encoded by a polynucleotide containing a base sequence having more than 90% identity with the base sequence of Serial Number 3 and having the same activity as CPT2.

[0069] (e) One or more polynucleotides selected from the polynucleotide sequence containing sequence number 11 and the polynucleotide encoding the protein described below, wherein the protein is encoded by a polynucleotide containing a base sequence having more than 90% identity with the base sequence of sequence number 11 and having the same activity as REF1.

[0070] (f) One or more polynucleotides selected from the polynucleotide sequence containing sequence number 13 and the polynucleotide encoding the protein described below, wherein the protein is encoded by a polynucleotide containing a base sequence having more than 90% identity with the base sequence of sequence number 13 and having the same activity as REF2.

[0071] (g) One or more polynucleotides selected from the polynucleotide sequence containing sequence number 15 and the polynucleotide encoding the protein described below, wherein the protein is encoded by a polynucleotide containing a base sequence having more than 90% identity with the base sequence of sequence number 15 and having the same activity as REF8, and

[0072] (h) One or more polynucleotides selected from the polynucleotide containing the base sequence of sequence number 17 and the polynucleotide encoding the protein described below, which is encoded by a polynucleotide containing a base sequence having more than 90% identity with the base sequence of sequence number 17 and having the same activity as SRPP1.

[0073]

[18] The cell according to any one of

[14] to

[17] , wherein the expression unit is a heterologous expression unit.

[0074]

[19] The cell according to

[14] contains the lipid membrane construct described in [9].

[0075]

[20] A method for preparing cells, wherein the cells are the cells described in

[14] .

[0076] The method includes a step of making the lipid membrane construct described in [9] interact with cells.

[0077]

[21] A method for manufacturing an isoprene polymer compound, comprising a step of performing an isoprene polymerization reaction using at least one of the compositions selected from any one of [1] to [6], the constructs selected from any one of [7] to [9], and the cells selected from any one of

[14] to

[19] .

[0078]

[22] According to the manufacturing method described in

[21] , isoprene polymerization is carried out using a low molecular weight allyl compound as a substrate.

[0079]

[23] A kit for manufacturing isoprene polymer compounds, comprising:

[0080] The composition selected from any one of [1] to [6], the construct selected from any one of [7] to [9], and at least one of the cells selected from any one of

[14] to

[19] , and a low molecular weight allyl compound.

[0081]

[24] A method for manufacturing rubber, comprising the step of manufacturing rubber using an isoprene polymer compound manufactured by the manufacturing method described in

[21] or

[22] .

[0082] Invention Effects

[0083] According to the present invention, natural rubber can be produced efficiently and stably, and a stable supply of rubber resources, which is expected to be in high demand globally, can be secured.

[0084] Detailed Implementation of the Invention

[0085] [Protein Composition]

[0086] (Isoprene polymerization activity)

[0087] The protein composition of the present invention exhibits isoprene polymerization activity. Isoprene polymerization activity generally refers to the activity that directly or indirectly promotes the polymerization reaction of isoprene to produce isoprene polymeric compounds. The polymerization reaction of isoprene is typically the polymerization reaction of isoprene in biological systems. Examples of substrates include low molecular weight allyl compounds such as isopentenyl diphosphate (IPP), farnesyl diphosphate (FPP), dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP), and geranylgeranyl diphosphate (GGPP).

[0088] As living organisms, examples include rubber trees (plants capable of producing natural rubber) such as plants of the genus Hevea (e.g., Brazilian rubber tree (Hevea brasiliensis), Hevea benthamiana, Hevea guianensis) and plants of the genus Ficus (e.g., Indian rubber tree (Ficus elastica), cypress rubber tree (Ficus lyrata), Benjamin rubber tree (Ficus benjamina), etc.), but are not limited to these, and also include other plant cells, animal cells, and microorganisms.

[0089] (Proteins that make up the protein composition)

[0090] (Proteins of (A) and (B))

[0091] The protein composition comprises at least one of proteins (A-1) and (A-2) and protein (B):

[0092] (A-1) One or more proteins selected from proteins containing the amino acid sequence of sequence number 6 and proteins containing an amino acid sequence that has more than 90% identity with the amino acid sequence of sequence number 6 and has the same activity as CPT6.

[0093] (A-2) One or more proteins selected from proteins containing the amino acid sequence of sequence number 8 and proteins containing an amino acid sequence having more than 90% identity with the amino acid sequence of sequence number 8 and having the same activity as CPT7; and

[0094] (B) One or more proteins selected from proteins containing the amino acid sequence of sequence number 10 and proteins containing an amino acid sequence that is more than 90% identical to the amino acid sequence of sequence number 10 and having the same activity as CPTL.

[0095] The amino acid sequences with sequence numbers 6, 8, and 10 are the amino acid sequences of cis-isopentenyltransferase (CPT) 6, CPT7, and CPTL from the Brazilian rubber tree (Hevea brasiliensis), respectively.

[0096] The proteins (A-1), (A-2), and (B) can be proteins encoded by the polynucleotides (a-1), (a-2), and (b) that encode their respective proteins:

[0097] (a-1) One or more polynucleotides selected from polynucleotides containing the base sequence of sequence number 5 and polynucleotides containing a base sequence having more than 90% identity with the base sequence of sequence number 5 and encoding a protein having the same activity as CPT6.

[0098] (a-2) One or more polynucleotides selected from the polynucleotide sequence containing sequence number 7 and polynucleotides containing a sequence having more than 90% identity with the sequence number 7 and encoding a protein having the same activity as CPT7; and

[0099] (b) One or more polynucleotides selected from the polynucleotides containing the base sequence of sequence number 9 and the polynucleotides encoding the following proteins, said proteins being encoded by polynucleotides containing a base sequence having more than 90% identity with the base sequence of sequence number 9 and having the same activity as CPTL.

[0100] The base sequences represented by sequence numbers 5, 7, and 9 are the full-length base sequences of CPT6, CPT7, and CPTL from the Brazilian rubber tree (Hevea brasiliensis).

[0101] (Other proteins)

[0102] The protein composition may contain proteins other than those of (A-1), (A-2), and (B). Examples of other proteins include proteins responsible for the polymerization of isoprene in vivo and proteins known to assist polymerization reactions. Examples of the former include the CPT family other than CPT6, CPT7, and CPTL. Examples of the latter include at least one protein selected from the rubber elongation factor (REF) family and the small rubberparticle protein (SRPP) family. Preferably, at least one protein selected from (C) to (H) is included.

[0103] (C) One or more proteins selected from proteins containing the amino acid sequence of sequence number 2 and proteins containing an amino acid sequence that has more than 90% identity with the amino acid sequence of sequence number 2 and has the same activity as CPT1.

[0104] (D) One or more proteins selected from proteins containing the amino acid sequence of sequence number 4 and proteins containing an amino acid sequence that has more than 90% identity with the amino acid sequence of sequence number 4 and has the same activity as CPT2.

[0105] (E) One or more proteins selected from proteins containing the amino acid sequence of sequence number 12 and proteins containing an amino acid sequence that is more than 90% identical to the amino acid sequence of sequence number 12 and having the same activity as REF1.

[0106] (F) One or more proteins selected from proteins containing the amino acid sequence of sequence number 14 and proteins containing an amino acid sequence that is more than 90% identical to the amino acid sequence of sequence number 14 and having the same activity as REF2.

[0107] (G) One or more proteins selected from proteins containing the amino acid sequence of sequence number 16 and proteins containing an amino acid sequence having more than 90% identity with the amino acid sequence of sequence number 16 and having the same activity as REF8; and

[0108] (H) One or more proteins selected from proteins containing the amino acid sequence of sequence number 18 and proteins containing an amino acid sequence that is more than 90% identical to the amino acid sequence of sequence number 18 and having the same activity as SRPP1.

[0109] The amino acid sequences of sequence numbers 2, 4, 12, 14, 16, and 18 contain mature proteins of CPT1, CPT2, REF1, REF2, REF8, and SRPP1 from the Brazilian rubber tree (Heveabrasiliensis), respectively.

[0110] Proteins (C) to (H) can be proteins encoded by polynucleotides (c) to (h) that encode their respective proteins:

[0111] (c) One or more polynucleotides selected from the polynucleotides containing the base sequence of Serial No. 1 and the polynucleotides encoding the following protein, said protein being encoded by a polynucleotide containing a base sequence having more than 90% identity with the base sequence of Serial No. 1 and having the same activity as CPT1.

[0112] (d) One or more polynucleotides selected from polynucleotides containing the base sequence of sequence number 3 and polynucleotides encoding the following protein, said protein being encoded by a polynucleotide containing a base sequence having more than 90% identity with the base sequence of sequence number 3 and having the same activity as CPT2.

[0113] (e) One or more polynucleotides selected from the polynucleotide containing the base sequence of sequence number 11 and the polynucleotide encoding the protein described below, which is encoded by a polynucleotide containing a base sequence having more than 90% identity with the base sequence of sequence number 11 and having the same activity as REF1.

[0114] (f) One or more polynucleotides selected from the polynucleotide containing the base sequence of sequence number 13 and the polynucleotide encoding the protein described below, which is encoded by a polynucleotide containing a base sequence having more than 90% identity with the base sequence of sequence number 13 and having the same activity as REF2.

[0115] (g) A polynucleotide selected from the polynucleotide sequence containing sequence number 15 and one or more polynucleotides encoding a protein that is encoded by a polynucleotide containing a base sequence having more than 90% identity with the base sequence of sequence number 15 and having the same activity as REF8; and

[0116] (h) One or more polynucleotides selected from the polynucleotide containing the base sequence of sequence number 17 and the polynucleotide encoding the protein described below, which is encoded by a polynucleotide containing a base sequence having more than 90% identity with the base sequence of sequence number 17 and having the same activity as SRPP1.

[0117] The base sequences represented by sequence numbers 1, 3, 11, 13, 15 and 17 are the full-length base sequences of CPT1, CPT2, REF1, REF2, REF8 and SRPP1 from the Brazilian rubber tree (Heveabrasiliensis).

[0118] The protein composition preferably includes two, three, four, or five proteins from (C) to (H), more preferably a combination of at least one of (C) and (D) and at least one of (E) to (H), and even more preferably all of them.

[0119] (identity%)

[0120] The identity of the amino acid and base sequences can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher. Identity can be determined using, for example, NCBI's BLAST (see http: / / www.ncbi.nlm.nih.gov) under default conditions.

[0121] (The activity possessed by the mutated protein)

[0122] "Having the same activity as each protein" means that, when measured under the same conditions, it has more than 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the activity of each protein.

[0123] (Mutations)

[0124] As long as the aforementioned proteins can maintain the same activity, variations can be introduced at sites within and outside the catalyst domain. The positions of the amino acid residues in the protein capable of maintaining the target activity can be specified by those skilled in the art. For example, those skilled in the art can 1) compare the amino acid sequences of multiple proteins with the same activity (e.g., the amino acid sequence represented by sequence number 6 or 8 and the amino acid sequences of other proteins related to isoprene polymerization), 2) identify relatively conserved or non-conserved regions, and then 3) be able to predict regions that play an important functional role and regions that do not, from the relatively conserved and non-conserved regions, thus identifying structural and / or functional correlations and specifying sites for the introduction of variations.

[0125] The mutation introduced into a protein can be a mutation in which an amino acid residue is replaced by another amino acid residue, preferably a mutation in which an amino acid residue with a similar side chain is replaced. Examples of amino acids classified based on amino acid residues with similar side chains include: amino acids with basic side chains such as lysine, arginine, and histidine; amino acids with acidic side chains such as aspartic acid and glutamic acid; amino acids with non-electrified polar side chains such as asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acids with non-polar side chains such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acids with β-branched side chains such as leucine, valine, and isoleucine; amino acids with aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine; amino acids with hydroxyl side chains (e.g., alcohols, phenoxy side chains) such as serine, threonine, and tyrosine; and amino acids with sulfur-containing side chains such as cysteine ​​and methionine.

[0126] For the purpose of codon optimization, base sequences can be altered. In this specification, codon optimization refers to altering a base sequence in a way that optimizes expression in a specific target cell without changing the sequence of the encoded polynucleotide. The optimized base sequence can be easily determined by anyone skilled in the art by understanding the frequency of codon usage in the introduced cell and utilizing the degeneracy of the genetic code. Examples of target cells include microorganisms such as Escherichia coli, plant cells, and animal cells.

[0127] (Protein modification)

[0128] Each protein constituting the protein composition may have a purification tag near its C-terminus. Examples of purification tags include, for instance, histidine tags; HA, FLAG, V5, and myc epitopes; chitin-binding protein (CBP); maltose-binding protein (MBP); glutathione-S-transferase (GST); and Strep tags. In addition to the above, the protein composition may also contain factors directly or indirectly related to the polymerization reaction of isoprene.

[0129] [Lipid Membrane Construct]

[0130] The lipid membrane construct of the present invention comprises a lipid membrane containing the above-described protein composition. By using the lipid membrane construct, the protein composition readily exhibits the polymerization activity of isoprene, enabling efficient isoprene polymerization.

[0131] Lipid membranes can be single-layered or bilayered. Examples of single-layered membrane constructs include rubber particles (particles extracted from latex from rubber trees). Examples of bilayered membrane constructs include lipoprotein bodies and artificial lipid membrane models. Artificial lipid membrane models can be composed of natural lipids, synthetic lipids, or both, without particular limitation. Bilayered membrane constructs are preferred, and lipoprotein bodies are more preferred.

[0132] (Lipoprotein bodies)

[0133] In this specification, a liposome is an artificial vesicle in which proteins are encapsulated or bound within a portion of the lipid bilayer membrane of a liposome. In this specification, a liposome is an artificial vesicle that captures aqueous components (usually water) within a space formed by being surrounded by a lipid bilayer membrane. The lipid bilayer membrane is typically composed of lipids such as phospholipids. Examples of phospholipids include phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, cardiolipin, or combinations thereof. Examples of lipids other than phospholipids include triacylglycerols, waxes, sphingolipids, and sterols and their fatty acid esters, or combinations thereof. The lipids constituting the lipid bilayer membrane preferably include phospholipids derived from organisms, and more preferably include phospholipids derived from soybeans.

[0134] The size of liposomes is not particularly limited, but their diameter is typically 50–300 nm. Liposomes can be naturally derived or synthetic, with the former being preferred, and more preferably derived from plants such as soybeans. Examples of synthetic liposomes include polyalkylene glycol-based liposomes.

[0135] The form in which the protein composition exists in the membrane lipid construct is not particularly limited. In the case of liposomes, it is presumed to be in the form of the protein composition bound to the hydrophilic group portion of the phospholipid on the surface of the liposome (a portion of which may be embedded inside the membrane).

[0136] (Manufacturing method)

[0137] Examples of methods for manufacturing membrane lipid constructs include, for instance, a method that involves expressing polynucleotides encoding proteins constituting the composition in a cell-free protein production system in the presence of a phospholipid-containing raw material.

[0138] The phospholipid-containing raw material includes liposomes when the membrane structure is a lipoprotein. As described above, liposomes of natural origin are preferred, especially those derived from plants such as soybeans.

[0139] The preferred method for manufacturing liposomes includes a step of expressing an expression unit containing the aforementioned polynucleotides in a cell-free protein production system in the presence of liposomes.

[0140] The expression unit contains at least one polynucleotide encoding each protein contained in the protein composition. The polynucleotide is a polynucleotide encoding a protein constituting the protein composition, including (a-1) or (a-2) and (b) above, and further including at least one of (c) to (h) as needed. The expression unit typically contains a promoter. Thus, the expression product of the polynucleotide encoded by the polynucleotide can be produced efficiently. Examples of promoters include the tac promoter, lac promoter, trp promoter, trc promoter, T7 promoter, T5 promoter, T3 promoter, and SP6 promoter, and may contain an enhancer. The promoter is attached to the polynucleotide in a manner that allows the polynucleotide to express itself, for example, attached upstream (5') of the coding sequence. The expression unit can be DNA or RNA, preferably DNA. The expression unit may contain factors such as a terminator, ribosome binding site, drug resistance gene, RNA processing signal, sequence to improve translation efficiency, sequence to improve protein stability, and sequence to improve protein secretion.

[0141] Cell-free protein production systems preferably use cell extracts (e.g., wheat germ extract, Escherichia coli extract, rabbit reticulocyte extract, insect cell extract) via dialysis multilayer method, which can be implemented according to commercially available cell-free protein expression kits.

[0142] Alternatively, other protein construction systems, such as artificial lipid membrane models, can be used instead of liposomes. The protein composition in an artificial lipid membrane model can be constructed using conventional methods. Furthermore, the artificial lipid membrane model can be composed of natural lipids, synthetic lipids, or both; there are no particular limitations.

[0143] (cell)

[0144] The cell of the present invention is a cell that expresses each of the proteins constituting the protein composition (proteins (A), (B), and at least one of (C) to (H) as needed). Examples of cells include, for example, microbial cells such as Escherichia coli and yeast, plant cells, and animal cells, but are not limited thereto.

[0145] The cell preferably contains an expression unit comprising the aforementioned polynucleotide. The expression unit is the same as described in the liposome description. The expression unit that the cell may contain is preferably a heterologous expression unit. In this specification, a heterologous expression unit refers to an expression unit in which at least one of the polynucleotide encoding the protein and the promoter typically included is not inherent to the host cell. Preferably, neither is inherent to the host cell.

[0146] As a method for manufacturing cells, one example is transformation using an expression vector containing an expression unit that encodes polynucleotides encoding each of the proteins constituting the protein composition described above. Since the protein composition, as described above, contains at least two proteins, the expression vector can be one (containing an expression unit with polynucleotides encoding all proteins) or two or more (containing expression units with individual or several polynucleotides). The expression vector can be an integrative vector or a non-integrative vector. In the expression vector, the polynucleotides can be configured under the control of a constitutive or inducible promoter.

[0147] Transformation of a host using an expression vector can be performed using more than one known method. Examples of such methods include the calcium phosphate method, liposome method, DEAE dextran method, electroporation, and particle gun (gene gun). Alternatively, phage vectors other than plasmid vectors can be used to infect bacteria and introduce the vector into the bacterial cell.

[0148] The cell can be a cell containing the aforementioned liposomes. The liposomes are preferably made from naturally occurring liposomes. This allows for easy interaction with the cell membrane. Cells containing liposomes can be manufactured by enabling this interaction. Methods of interaction include, for example, adsorbing or binding the liposomes to the cell surface, allowing the liposomes to enter the cell (endocytosis or phagocytosis), and fusing the lipid bilayer membrane of the liposomes with the cell membrane.

[0149] [Method for manufacturing isoprene polymer compounds]

[0150] The above-mentioned protein composition, lipoprotein bodies, and cells can be used in the manufacture of isoprene polymer compounds.

[0151] In the manufacture of isoprene polymers, an isoprene polymerization reaction is carried out using a low molecular weight allyl compound as a substrate. Examples of low molecular weight allyl compounds include isopentenyl diphosphate and farnesyl diphosphate, with both being preferred. Reaction temperature, reaction time, and other conditions can be appropriately set. Cis-polyisoprene is a representative isoprene polymer, useful as a raw material for rubber. Other examples of isoprene polymers include polyols, polyterpenoids, and their derivatives, which are useful as materials for pharmaceuticals and health foods due to their physiological activities in living organisms (e.g., immune activity, antiviral activity, antioxidant activity). Furthermore, they can also be used as additives (e.g., plasticizers, compatibilizers) in rubber products.

[0152] [Reagent kit for the manufacture of isoprene polymer compounds]

[0153] The aforementioned protein composition, liposomes, and cells can be used as a kit for manufacturing isoprene polymer compounds. The kit typically contains a low molecular weight allyl compound as a substrate. Furthermore, the reaction vessel can contain the desired reagents.

[0154] [Rubber Manufacturing Methods]

[0155] The isoprene polymer compound obtained by the above-described method can be used in the manufacture of rubber.

[0156] The steps and conditions for manufacturing rubber from isoprene polymers can be followed using conventional methods, such as cross-linking and molding the isoprene polymers after mixing. Additives that can be used as needed include, for example, reinforcing agents, silane coupling agents, fillers, vulcanizing agents, vulcanization accelerators, vulcanization aids, oils, curing resins, waxes, anti-aging agents, and colorants. These additives are added at the appropriate stages. The resulting rubber can be used in various applications such as tires, building materials, sporting goods, and automotive parts. Example

[0157] Examples 1-11

[0158] (1) Obtaining the genes for biosynthetic proteins in natural rubber

[0159] Seven proteins (CPT1, CPT2, CPTL, REF1, REF2, REF8, and SRPP1) that are highly produced in latex were selected from the Rubber Database (http: / / Matsui-lab.riken.jp / rubber / home.html). In addition, two proteins (CPT6 and CPT7) were selected from phylogenetic analysis.

[0160] The CPT1, CPT2, CPT6, CPT7, CPTL, REF1, REF2, REF8, and SRPP1 genes were obtained by PCR using cDNA encoding the genes of each protein or plasmids containing the genes as templates. PCR was performed according to the instructions of PrimeSTAR Max DNA Polymerase (Takara) or KOD One PCR Master Mix (TOYOBO), with restriction enzyme digestion sites and His tag sequences added to the primers as needed.

[0161] The primer sets used to obtain the CPT1, CPT2, CPT6, CPT7, CPTL, REF1, REF2, REF8, and SRPP1 genes respectively are primers 1 and 2, primers 3 and 4, primers 5 and 6, primers 7 and 8, primers 9 and 10, primers 11 and 12, primers 13 and 14, primers 15 and 16, and primers 17 and 18.

[0162] (Primer set used to obtain the CPT1 gene)

[0163] Primer 1: 5'-atggaattatacaacggtg-3' (serial number 19)

[0164] Primer 2: 5'-atctcgagttaatgatgatgatgatgatgttttaag-3' (Sequence No. 20)

[0165] (Primer set used to obtain the CPT2 gene)

[0166] Primer 3: 5'-atggaaatatatacgggtcag-3' (Sequence No. 21)

[0167] Primer 4: 5'-atctcgagttaatgatgatgatgatgatgttttaaatattc-3' (Sequence No. 22)

[0168] (Primer set used to obtain the CPT6 gene)

[0169] Primer 5: 5'-atggaaaaacatagcagtag-3' (Sequence No. 23)

[0170] Primer 6: 5'-atctcgagttatataactgatgctttttc-3' (Sequence No. 24)

[0171] (Primer set used to obtain the CPT7 gene)

[0172] Primer 7: 5'-atgcaatccttgcacttg-3' (Sequence No. 25)

[0173] Primer 8: 5'-atctcgagttatgtaagtcgtctaccatag-3' (Sequence No. 26)

[0174] (Primer set used to obtain the CPTL gene)

[0175] Primer 9: 5'-atggatttgaaacctgg-3' (Sequence No. 27)

[0176] Primer 10: 5'-atctcgagttaatgatgatgatgatgatgtgtac-3' (Sequence No. 28)

[0177] (Primer set used to obtain the REF1 gene)

[0178] Primer 11: 5'-atggctgaaggtgaagaagaggtgaatatc-3' (Sequence No. 29)

[0179] Primer 12: 5'-atctcgagtcacccatctccatatagcac-3' (Sequence No. 30)

[0180] (Primer set used to obtain the REF2 gene)

[0181] Primer 13: 5'-atggctgaagacgaagacaaccaacaag-3' (Sequence No. 31)

[0182] Primer 14: 5'-atctcgagtcaattctctccataaaacac-3' (Sequence No. 32)

[0183] (Primer set used to obtain the REF8 gene)

[0184] Primer 15: 5'-atggctgaagggaaagaaaacgagaatttc-3' (Sequence No. 33)

[0185] Primer 16: 5'-atctcgagttactctgcactttccttcac-3' (Sequence No. 34)

[0186] (Primer set used to obtain the SRPP1 gene)

[0187] Primer 17: 5'-atggctgaagaggtggaggaagagaggc-3' (Sequence No. 35)

[0188] Primer 18: 5'-atctcgagttatgatgcctcatctccaaac-3' (Sequence No. 36)

[0189] For each gene obtained by the above method, its sequence was identified, including the full-length base sequence and amino acid sequence. The base sequences of CPT1, CPT2, CPT6, CPT7, CPTL, REF1, REF2, REF8, and SRPP1 genes are shown in sequence numbers 1, 3, 5, 7, 9, 11, 13, 15, and 17, respectively, and the amino acid sequences are shown in sequence numbers 2, 4, 6, 8, 10, 12, 14, 16, and 18, respectively.

[0190] (2) Preparation of plasmids for cell-free protein synthesis

[0191] After the PCR reaction products of each gene obtained in (1) above were treated with the restriction enzyme XhoI attached to the primers, the combinations in Table 1 were selected and inserted into the cell-free expression vector pEU-E01-MCS treated with EcoRV and XhoI restriction enzymes, thereby preparing a cell-free protein synthesis plasmid.

[0192] (3) Transformation of Escherichia coli

[0193] The plasmid prepared above was transformed into Escherichia coli JM109, and the transformants were cultured on LB agar medium containing ampicillin. The transformants that had been introduced with the target plasmid were selected by colony PCR.

[0194] (4) Preparation of plasmids for cell-free protein synthesis

[0195] E. coli transformed with a plasmid containing the target gene were cultured overnight on LB liquid medium at 37°C. The bacterial cells were then recovered, and the plasmid was recovered. Plasmid recovery was performed using the FastGene Plasmid Mini Kit (Genetics, Japan). Sequencing analysis confirmed that the nucleotide sequence of the gene inserted into the recovered plasmid was unchanged.

[0196] (5) Transformation of Escherichia coli

[0197] The plasmids prepared above were used to transform Escherichia coli JM109, and the transformants were cultured on LB agar medium containing ampicillin.

[0198] (6) Recovery of plasmids used in cell-free protein synthesis

[0199] After transforming E. coli with a plasmid containing the target gene and culturing it overnight at 37°C on LB liquid medium, the bacterial cells were recovered, and the plasmid was recovered. Plasmid recovery was performed using the QIAGEN Plasmid Midi Kit (QIAGEN), adjusted to 1 μg / μL.

[0200] (7) Synthesis of membrane proteins (liposomes) in the presence of liposomes using multilayer dialysis

[0201] Lipid bilayer membranes (liposomes: approximately several hundred nm in diameter) were prepared using phospholipids derived from soybean. Cell-free protein synthesis was performed under liposome-added conditions according to the protocol provided with the ProteoLiposome BD Expression Kit (CellFreeSciences). For the nine protein expression plasmids serving as templates, the plasmids were adjusted to 1 μg / μL for use in the combinations shown in Table 1, and used in equal proportions to ensure a total volume of approximately 13 μL. For example, in Example 3, 1.5 μL of each of the nine proteins was added to the transcription reaction system, totaling 13.5 μL. After the synthesis reaction, the liposomes containing the nine proteins were simply purified according to the provided protocol, and the synthesis was confirmed by SDS-PAGE.

[0202] (8) Determination of isoprene polymerization activity

[0203] The isoprene polymerization activity of cell-free synthesized proteins was determined by the following method.

[0204] The following solutions were prepared: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 2 mM DTT, 10 μM farnesyl diphosphate (FPP), and 50 μM... 14 C-Isopentenyl diphosphate ( 14 C-IPP (specific radioactivity: 1.48-2.22 GBq / mmol), 100 μL of a reaction solution containing 5 μL of lipoprotein bodies, were reacted at 30°C for 24 hours.

[0205] After the reaction, 200 μL of saturated saline was added, and the reaction product was extracted with 300 μL of water-saturated n-butanol. 400 μL of 2M NaCl was added to the recovered water-saturated n-butanol layer, and after resuspending, the water-saturated n-butanol layer was recovered. 5 mL of Nacalai Tesque was added to 50 μL of the recovered solution, and after thorough mixing, the radiation dose (dpm) was measured using a liquid scintillation counter (LSC-8000; Aloka). The reaction system without liposomes was used as a negative control. The results using liposomes containing various proteins are shown in Table 1.

[0206] (9) TLC images of the products of isoprene polymerization reaction

[0207] The water-saturated n-butanol solution recovered above was used to evaporate the organic layer using a centrifugal evaporator. 1.5 mL of a reaction solution containing a final concentration of 100 mM sodium acetate (pH 5.5), a final concentration of 1 U acid phosphatase, and 1 mL of methanol from potatoes was added, and the mixture was reacted at 37°C for 17 hours.

[0208] After the reaction, the sample was extracted with 500 μL of hexane, and the resulting hexane layer was concentrated and dried using a centrifugal evaporator. The dried product was redissolved with 10 μL of hexane, and the total volume was spotted onto a TLC plate (Merck, TLC glass plate RP-18F254S 5×10 cm). After development with acetone:water = 19:1, the sample was exposed on an imaging plate and visualized using a scanner (Typhoon FLA9500).

[0209] Comparative Example 1 (Negative Control Group)

[0210] Perform the same procedures as in the above examples, except without adding lipoprotein bodies.

[0211] Comparative Examples 2–9 (excluding the negative control group)

[0212] The plasmids for cell-free synthesis of each protein were adjusted to 1 μg / μL. The total amount of plasmids in the reaction was set to 13.5 μL, and each plasmid was added in equal proportions. The same procedures as in the previous examples were then performed.

[0213] [Table 1]

[0214] Table 1. Results of protein combinations and radiation dose measurements for each embodiment.

[0215]

[0216] As clearly shown in Table 1, the radiation doses of each embodiment were higher than those of the comparative examples, and products (presumably with a molecular weight of around 1000-2000) were synthesized. The radiation doses of Example 1 (a combination of CPT6 and CPTL) and Example 2 (a combination of CPT7 and CPTL) were higher than those of the comparative examples, thus confirming that each combination exhibited isoprene polymerization activity. Furthermore, the radiation doses of Examples 3-11, which further incorporated other proteins, were also higher than those of the comparative examples, and also higher than those of Examples 1 and 2, thus confirming the possibility that proteins other than CPT6, 7, and CPTL may also promote isoprene polymerization activity.

[0217] The above results demonstrate that natural rubber can be produced efficiently using this invention.

[0218] [Sequence List Free Text]

[0219] Serial number 1: Base sequence of CPT1

[0220] Serial number 2: Amino acid sequence of CPT1

[0221] Serial number 3: Base sequence of CPT2

[0222] Serial number 4: Amino acid sequence of CPT2

[0223] Serial number 5: Base sequence of CPT6

[0224] Serial number 6: Amino acid sequence of CPT6

[0225] Serial number 7: Base sequence of CPT7

[0226] Serial number 8: Amino acid sequence of CPT7

[0227] Serial number 9: Base sequence of CPTL

[0228] Serial number 10: Amino acid sequence of CPTL

[0229] Serial number 11: Base sequence of REF1

[0230] Serial number 12: Amino acid sequence of REF1

[0231] Serial number 13: Base sequence of REF2

[0232] Serial number 14: Amino acid sequence of REF2

[0233] Serial number 15: Base sequence of REF8

[0234] Serial number 16: amino acid sequence of REF8

[0235] Serial number 17: Base sequence of SRPP1

[0236] Serial number 18: Amino acid sequence of SRPP1

[0237] Serial number 19: Primer 1 (used to obtain the CPT1 gene)

[0238] Sequence number 20: Primer 2 (used to obtain the CPT1 gene)

[0239] Serial number 21: Primer 3 (used to obtain the CPT2 gene)

[0240] Sequence number 22: Primer 4 (used to obtain the CPT2 gene)

[0241] Sequence number 23: Primer 5 (used to obtain the CPT6 gene)

[0242] Sequence number 24: Primer 6 (used to obtain the CPT6 gene)

[0243] Sequence number 25: Primer 7 (used to obtain the CPT7 gene)

[0244] Sequence number 26: Primer 8 (used to obtain the CPT7 gene)

[0245] Serial number 27: Primer 9 (used to obtain the CPTL gene)

[0246] Serial number 28: Primer 10 (used to obtain the CPTL gene)

[0247] Serial number 29: Primer 11 (used to obtain the REF1 gene)

[0248] Sequence number 30: Primer 12 (used to obtain the REF1 gene)

[0249] Sequence number 31: Primer 13 (used to obtain the REF2 gene)

[0250] Sequence number 32: Primer 14 (used to obtain the REF2 gene)

[0251] Sequence number 33: Primer 15 (used to obtain the REF8 gene)

[0252] Sequence number 34: Primer 16 (used to obtain the REF8 gene)

[0253] Sequence number 35: Primer 17 (used to obtain the SRPP1 gene)

[0254] Sequence number 36: Primer 18 (used to obtain the SRPP1 gene). sequence list <110> Sumitomo Riko Co., Ltd. <120> Protein compositions with isoprene polymerization activity <130> PA-R032779 <150> JP 2020-183088 <151> 2020-10-30 <160> 36 <170> PatentIn version 3.5 <210> 1 <211> 873 <212> DNA <213> Brazilian rubber tree (Hevea brasiliensis) <400> 1 atggaattat acaacggtga gaggccaagt gtgttcagac ttttagggaa gtatatgaga 60 aaagggttat atagcatcct aacccagggt cccatcccta ctcatattgc cttcatattg 120 gatggaaaca ggaggtttgc taagaagcat aaactgccag aaggaggtgg tcataaggct 180 ggatttttag ctcttctgaa cgtactaact tattgctatg agttaggagt gaaatatgcg 240 actatctatg cctttagcat cgataatttt cgaaggaaac ctcatgaggt tcagtacgta 300 atggatctaa tgctggagaa gattgaaggg atgatcatgg aagaaagtat catcaatgca 360 tatgatattt gcgtacgttt tgtgggtaac ctgaagcttt taagtgagcc agtcaagacc 420 gcagcagata agattatgag ggctactgcc aacaattcca aatgtgtgct tctcattgct 480 gtatgctata cttcaactga tgagatcgtg catgctgttg aagaatcctc tgaattgaac 540 tccaatgaag tttgtaacaa tcaagaattg gaggaggcaa atgcaactgg aagcggtact 600 gtgattcaaa ttgagaacat ggagtcgtat tctggaataa aacttgtaga ccttgagaaa 660 aacacctaca taaatcctta tcctgatgtt ctgattcgaa cttctgggga gacccgtctg 720 agcaactact tactttggca gactactaat tgcatactgt attctcctca tgcactgtgg 780 ccagagattg gtcttcgaca cgtgtgtgg gcagtaatta actgccaacg tcattattct 840 tacttggaga aacataagga atacttaaaa 873 <210> 2 <211> 290 <212> PRT <213> Hevea brasiliensis (Hevea brasiliensis) <400> 2 Met Glu Leu Tyr Asn Gly Glu Arg Ser Val Phe Arg Leu Leu Gly 1 5 10 15 Lys Tyr Met Arg Lys Gly To Tyr Ser Ile To Thr Gln Gly Pro Ile 20 25 30 Pro Thr His Ile Ala Phe Ile Leu Asp Gly Asn Arg Arg Phe Ala Lys 35 40 45 Lys His Lys Leu Pro Glu Gly Gly Gly His Lys Ala Gly Phe Leu Ala 50 55 60 Mice Asn Val Thr Tyr Cys Tyr Glu Mice Gly Val Lys Tyr Ala 65 70 75 80 Thr Ile Tyr Ala Phe Ser Ile Asp Asn Phe Arg Arg Lys Pro His Glu 85 90 95 Val Gln Tyr Val Met Asp Leu Met Leu Glu Lys Ile Glu Gly Met Ile 100 105 110 Met Glu Glu Ser Ile Ile Asn Ala Tyr Asp Ile Cys Val Arg Phe Val 115 120 125 Gly Asn Leu Lys Leu Leu Ser Glu Pro Val Lys Thr Ala Ala Asp Lys 130 135 140 Ile Met Arg Ala Thr Ala Asn Asn Ser Lys Cys Val Leu Leu Ile Ala 145 150 155 160 Val Cys Tyr Thr Ser Thr Asp Glu Ile Val His Ala Val Glu Glu Ser 165 170 175 Ser Glu Leu Asn Ser Asn Glu Val Cys Asn Asn Gln Glu Leu Glu Glu 180 185 190 Ala Asn Ala Thr Gly Ser Gly Thr Val Ile Gln Ile Glu Asn Met Glu 195 200 205 Ser Tyr Ser Gly Ile Lys Leu Val Asp Leu Glu Lys Asn Thr Tyr Ile 210 215 220 Asn Pro Tyr Pro Asp Val Leu Ile Arg Thr Ser Gly Glu Thr Arg Leu 225 230 235 240 Ser Asn Tyr Leu Leu Trp Gln Thr Thr Asn Cys Ile Leu Tyr Ser Pro 245 250 255 His Ala Leu Trp Pro Glu Ile Gly Leu Arg His Val Val Trp Ala Val 260 265 270 Ile Asn Cys Gln Arg His Tyr Ser Tyr Leu Glu Lys His Lys Glu Tyr 275 280 285 Leu Lys 290 <210> 3 <211> 891 <212> DNA <213> Hevea brasiliensis <400> 3 atggaaatat atacgggtca gaggccaagt gtgtttagaa ttttgggaa atacatgaga 60 aaagggttat atagcatcct aacccaaggt cccatcccta ctcatcttgc cttcataatg 120 gatggaaacc ggaggttgc taagaagcac aaaatgaaag aagcagaagg ttataaggca 180 ggatatttag ctcttctgag aacactaact tattgctatg agttgggagt gaggtatgta 240 accatttatg cctttagcat tgataatttt cgaaggcaac ctcgtgaggt tcagtgcgta 300 atgaatctaa tgatggagaa gattgaagag attatcgtgg aagaaagtat catgaatgca 360 tatgatgttg gcgtacgtat tgtgggtaac ctgaatcttt tagatgagcc aatcaggatc 420 gcagcagaaa agattatgag ggctactgcc aataattcca ggtttgtgct tctcattgct 480 gtagcctata gttcaactga tgagatcgtg catgctgttg aagaatcctc taaagacaaa 540 ttgaactcca atgaagtttg caacaatggg attgaagctg aacaagaatt taaggaggca 600 aacggaactg gaaacagtgt gattccagtt cagaagacgg agtcatattc tggaataaat 660 cttgcagacc ttgagaaaaa cacctacgta aatcctcatc ctgatgtctt gattcgaact 720 tctgggttga gccgtctaag taactaccta ctttggcaga ctagtaattg catactgtat 780 tctccttttg cactgtggcc agagattggt ctcaggcact tggtatggac agtaatgaac 840 ttccaacgtc atcattctta tttggagaag cataaggaat attaaaata a 891 <210> 4 <211> 296 <212> PRT <213> Hevea brasiliensis <400> 4 Met Glu Ile Tyr Thr Gly Gln Arg Pro Ser Val Phe Arg Ile Phe Gly 1 5 10 15 Lys Tyr Met Arg Lys Gly Leu Tyr Ser Ile Leu Thr Gln Gly Pro Ile 20 25 30 Pro Thr His Leu Ala Phe Ile Met Asp Gly Asn Arg Arg Phe Ala Lys 35 40 45 Lys His Lys Met Lys Glu Ala Glu Gly Tyr Lys Ala Gly Tyr Leu Ala 50 55 60 Leu Leu Arg Thr Leu Thr Tyr Cys Tyr Glu Leu Gly Val Arg Tyr Val 65 70 75 80 Thr Ile Tyr Ala Phe Ser Ile Asp Asn Phe Arg Arg Gln Pro Arg Glu 85 90 95 Val Gln Cys Val Met Asn Leu Met Met Glu Lys Ile Glu Glu Ile Ile 100 105 110 Val Glu Glu Ser Ile Met Asn Ala Tyr Asp Val Gly Val Arg Ile Val 115 120 125 Gly Asn Leu Asn Leu Leu Asp Glu Pro Ile Arg Ile Ala Ala Glu Lys 130 135 140 Ile Met Arg Ala Thr Ala Asn Asn Ser Arg Phe Val Leu Leu Ile Ala 145 150 155 160 Val Ala Tyr Ser Ser Thr Asp Glu Ile Val His Ala Val Glu Glu Ser 165 170 175 Ser Lys Asp Lys Leu Asn Ser Asn Glu Val Cys Asn Asn Gly Ile Glu 180 185 190 Ala Glu Gln Glu Phe Lys Glu Ala Asn Gly Thr Gly Asn Ser Val Ile 195 200 205 Pro Val Gln Lys Thr Glu Ser Tyr Ser Gly Ile Asn Leu Ala Asp Leu 210 215 220 Glu Lys Asn Thr Tyr Val Asn Pro His Pro Asp Val Leu Ile Arg Thr 225 230 235 240 Ser Gly Leu Ser Arg Leu Ser Asn Tyr Leu Leu Trp Gln Thr Ser Asn 245 250 255 Cys Ile Leu Tyr Ser Pro Phe Ala Leu Trp Pro Glu Ile Gly Leu Arg 260 265 270 His Leu Val Trp Thr Val Met Asn Phe Gln Arg His His Ser Tyr Leu 275 280 285 Glu Lys His Lys Glu Tyr Leu Lys 290 295 <210> 5 <211> 1122 <212> DNA <213> Hevea brasiliensis <400> 5 atggaaaaac atagcagtag tagagtgagt gagctgtttg gaaatttggg tagttttatt 60 agaatttgca tatttcgtgt gttatccatg ggacccatcc ccaatcattt tgccttcata 120 atggatggaa atcggaggta tgctaagaag gagaacatga aaaaaggggc aggtcatagg 180 gctggattct tagctcttat atccatactt aagtactgct atgaattggg agtgaagtat 240 gtaacaattt acgcctttag cattgataat ttcaaaagga gtcctgatga agttaaggac ctgatggatc tgatgctaga aaagattgag gatttgctga gggacgaaag cattgtgaac caatatgga tcagagtata ttttataggt aatttgaaac ttttgagtga aactgtgagg attgcagcag aaaaggttat gaaagctact gccaaaaaca ccaattgtac ccttttaatc tgcgtagcct atacgtcacg tgatgagatt gtacatgctg ttcaagtttc atgtaaaaat aaacaggagg aaattcaacc attgagcttt tgtaaagcta aatgatgc cattgaagaa gtagaggata ataagaaggt taatggagtc atcccatttg tttttttaga atcccagaaa gatgaagcag gcaaatctca agcaacaatg gcaagtgtaa cctgcagttg tctggctaga ggagttgaag ggggtggcaa caaaatagc atggttgttc gtgctgtccg aggatcctat 780 gaagataaat gggataactg tcaagcaatg atggaaaata gaactggcaa tggtgtgact ttatctgaag agtagtgaag tatgcaggga gagtgttcta tcataaagct agtagcatt gagaaacaga tgtacatggc tgtagctcct gaacctgaca tccttattcg aagttctgga gagtcccgcc tgagtactt cctactttgg cagaccagtg agtgccagtt atattctcca 1020 gatgcattgt ggccagaaat tggtttatgg cacttggtgt gggcagtatt aaacttccaa 1080 cgaaaccatg cttatttgga aagaaaaag catcagttat aa 1122 <210> 6 <211> 373 <212> PRT <213> Hevea brasiliensis (Hevea brasiliensis) <400> 6 Met Glu Lys His Ser Ser Ser Arg Val Ser Glu Leu Phe Gly Asn Leu 1 5 10 15 Gly Ser Phe Ile Arg Ile Cys Ile Phe Arg Val Leu Ser Met Gly Pro 20 25 30 Ile Pro Asn His Phe Ala Phe Ile Met Asp Gly Asn Arg Arg Tyr Ala 35 40 45 Lys Lys Glu Asn Met Lys Lys Gly Ala Gly His Arg Ala Gly Phe Leu 50 55 60 Tyr Cys Tyr Glu Gly Val Lys Tyr 65 70 75 80 Val Thr Ile Tyr Ala Phe Ser Ile Asp Phe Lys Arg Ser Pro Asp 85 90 95 Glu Val Lys Asp Leu Met Asp Leu Met Leu Glu Lys Ile Glu Asp Leu 100 105 110 Leu Arg Asp Glu Ser Ile Val Asn Gln Tyr Gly Ile Arg Val Tyr Phe 115 120 125 Ile Gly Asn Leu Lys Leu Leu Ser Glu Thr Val Arg Ile Ala Ala Glu 130 135 140 Lys Val Met Lys Ala Thr Ala Lys Asn Thr Asn Cys Thr Leu Leu Ile 145 150 155 160 Cys Val Ala Tyr Thr Ser Arg Asp Glu Ile Val His Ala Val Gln Val 165 170 175 Ser Cys Lys Asn Lys Gln Glu Glu Ile Gln Pro Leu Ser Phe Cys Lys 180 185 190 Ala Asn Asn Asp Ala Ile Glu Glu Val Glu Asp Asn Lys Lys Val Asn 195 200 205 Gly Val Ile Pro Phe Val Phe Leu Glu Ser Gln Lys Asp Glu Ala Gly 210 215 220 Lys Ser Gln Ala Thr Met Ala Ser Val Thr Cys Ser Cys Leu Ala Arg 225 230 235 240 Gly Val Glu Gly Gly Gly Asn Lys Asn Ser Met Val Val Arg Ala Val 245 250 255 Arg Gly Ser Tyr Glu Asp Lys Trp Asp Asn Cys Gln Ala Met Met Glu 260 265 270 Asn Arg Thr Gly Asn Gly Val Thr Leu Ser Glu Glu Ser Glu Asn Met 275 280 285 Gln Gly Glu Cys Ser Ile Ile Lys Leu Val Asp Ile Glu Lys Gln Met 290 295 300 Tyr Met Ala Val Ala Pro Glu Pro Asp Ile Leu Ile Arg Ser Ser Gly 305 310 315 320 Glu Ser Arg Leu Ser Asn Phe Leu Leu Trp Gln Thr Ser Glu Cys Gln 325 330 335 Leu Tyr Ser Pro Asp Ala Leu Trp Pro Glu Ile Gly Leu Trp His Leu 340 345 350 Val Trp Ala Val Leu Asn Phe Gln Arg Asn His Ala Tyr Leu Glu Lys 355 360 365 Lys Lys His Gln Leu 370 <210> 7 <211> 939 <212> DNA <213> Hevea brasiliensis <400> 7 atgcaatcct tgcacttggc cctccctctc cctttttaca acactctaac tcctcaaaaa 60 cacaaacact tcctttctcc cgatgccaga actaatacac gcccaattca ccgccttctc 120 ggggccaaaa cagacgtagc tgtcaaacag gaggaaacta ttgctgtaag caccggagac 180 tcgcccaaga ctgaaccttt gccggagggg ctccggggag agttgatgcc acggcatgtc 240 gccgtgatca tggacggcaa tgggaggtgg gcccagctgc gaggtcagat ggcatcgatg 300 ggtcatcagg ctggtgcacg gtctttgctg gagatcgtgc agctttcttg tcaatggggg 360 attaaagttc ttagcgtttt tgcgttttct tgcgataatt ggactaggcc caaagtcgtg 420 caggtggaga ttgatttctt gatgagtttg ttcgaaagcg tgttaaagtc agagatggat 480 aaatttgtga gggaaggtat tcgaatctct gtgatcgggg actcatcaag gcttccacag 540 tctttgcaaa gattaataaa tgaagtggag gagaccacca gaaatttctc gaaactgcac 600 cttctagtgg cggttagcta cagtggaaag tatgatgttg taaaagcatg caaaagtatt 660 gcttgtctgg taaaggatgg tgttattgaa ccagaagaca ttagcgaaag cctaattgag 720 caggagttgg aaacaaattg ctccgagttt ccctcccctg atttattaat ccgaactagt 780 ggtgaactta gaattagcaa cttcttgcta tggcagttgg cctacactga acttttcttt 840 gcggaagaac tctggcctga ttttggaaaa actggattca tagaggcctt aacttcatac 900 caacaaaggc aaagacgcta tggtagacga cttacataa 939 <210> 8 <211> 312 <212> PRT <213> Hevea brasiliensis <400> 8 Put Gln Ser Leu His Leu Ala Leu Pro Leu Pro Phe Tyr Asn Thr Leu 1 5 10 15 Thr Pro Gln Lys His Lys His Phe Leu Ser Pro Asp Ala Arg Thr Asn 20 25 30 Thr Arg Pro Ile His Arg Leu Leu Gly Ala Lys Thr Asp Val Ala Val 35 40 45 Lys Gln Glu Glu Thr Ile Ala Val Ser Thr Gly Asp Ser Pro Lys Thr 50 55 60 Glu Pro Leu Pro Glu Gly Leu Arg Gly Glu Leu Met Pro Arg His Val 65 70 75 80 Ala Val Ile Met Asp Gly Asn Gly Arg Trp Ala Gln Leu Arg Gly Gln 85 90 95 Put Ala Ser Put Gly His Gln Ala Gly Ala Arg Ser Leu Leu Glu Ile 100 105 110 Val Gln Leu Ser Cys Gln Trp Gly Ile Lys Val Leu Ser Val Phe Ala 115 120 125 Phe Ser Cys Asp Asn Trp Thr Arg Pro Lys Val Val Gln Val Glu Ile 130 135 140 Asp Phe Leu Met Ser Leu Phe Glu Ser Val Leu Lys Ser Glu Met Asp 145 150 155 160 Lys Phe Val Arg Glu Gly Ile Arg Ile Ser Val Ile Gly Asp Ser Ser 165 170 175 Arg Leu Pro Gln Ser Leu Gln Arg Leu Ile Asn Glu Val Glu Glu Thr 180 185 190 Thr Arg Asn Phe Ser Lys Leu His Leu Leu Val Ala Val Ser Tyr Ser 195 200 205 Gly Lys Tyr Asp Val Val Lys Ala Cys Lys Ser Ile Ala Cys Leu Val 210 215 220 Lys Asp Gly Val Ile Glu Pro Glu Asp Ile Ser Glu Ser Leu Ile Glu 225 230 235 240 Gln Glu Leu Glu Thr Asn Cys Ser Glu Phe Pro Ser Pro Asp Leu Leu 245 250 255 Ile Arg Thr Ser Gly Glu Leu Arg Ile Ser Asn Phe Leu Leu Trp Gln 260 265 270 Leu Ala Tyr Thr Glu Leu Phe Phe Ala Glu Glu Leu Trp Pro Asp Phe 275 280 285 Gly Lys Thr Gly Phe Ile Glu Ala Leu Thr Ser Tyr Gln Gln Arg Gln 290 295 300 Arg Arg Tyr Gly Arg Arg Leu Thr 305 310 <210> 9 <211> 774 <212> DNA <213> Hevea brasiliensis <400> 9 atggatttga aacctggagc tggagggcag agagttaatc gtttagtgga tccgattagt 60 tatcattttc ttcaatttct gtggcgtact ctacatcttc ttgtcagctt atggtacctt 120 caagttagta tggtccaaat gatcgaaggc tttctaatct ctagtggact tgtgaaacgc 180 tatggagccc tcgatattga caaggtccgg taccttgcca ttgtggtaga tagtgaagaa 240 gcttaccaaa tttctaaagt tattcagctt ttgaaatggg tggaagatat gggtgtgaaa 300 catttatgcc tctatgattc aaaaggagtt ctcaagacaa acaagaaaac catcatggag 360 agtttgaaca atgctatgcc atttgaggaa gcagttgaaa aagatgtttt actggaccag 420 aaacagatga ctgtggaatt tgcttccagc tccgatggaa aggaagcaat aaccagggca 480 gctaacgtac tctttatgaa gtatttgaag tatgctaaaa ctggtgtagg aaaggaagaa 540 ccatgcttta cagaagatca aatggatgag gcactaaaag ctataggtta caaagggccg 600 gaacctgact tgctattaat tttggacct gttagatgcc atctaggttt ctcaccgtgg 660 agacttcgat atactgagat ggtgcatatg ggacccttga ggtacatgaa cctcggttca 720 ctaaaaaagg ccattcacag gttcacaaca gtgcagcaaa attatggtac ataa 774 <210> 10 <211> 257 <212> PRT <213> Hevea brasiliensis <400> 10 Put Asp Leu Lys Pro Gly Ala Gly Gly Gln Arg Val Asn Arg Leu Val 1 5 10 15 Asp Pro Ile Ser Tyr His Phe Leu Gln Phe Leu Trp Arg Thr Leu His 20 25 30 Leu Leu Val Ser Leu Trp Tyr Leu Gln Val Ser Met Val Gln Met Ile 35 40 45 Glu Gly Phe Leu Ile Ser Ser Gly Leu Val Lys Arg Tyr Gly Ala Leu 50 55 60 Asp Ile Asp Lys Val Arg Tyr Leu Ala Ile Val Val Asp Ser Glu Glu 65 70 75 80 Ala Tyr Gln Ile Ser Lys Val Ile Gln Leu Leu Lys Trp Val Glu Asp 85 90 95 Met Gly Val Lys His Leu Cys Leu Tyr Asp Ser Lys Gly Val Leu Lys 100 105 110 Thr Asn Lys Lys Thr Ile Met Glu Ser Leu Asn Asn Ala Met Pro Phe 115 120 125 Glu Glu Ala Val Glu Lys Asp Val Leu Leu Asp Gln Lys Gln Met Thr 130 135 140 Val Glu Phe Ala Ser Ser Ser Asp Gly Lys Glu Ala Ile Thr Arg Ala 145 150 155 160 Ala Asn Val Leu Phe Met Lys Tyr Leu Lys Tyr Ala Lys Thr Gly Val 165 170 175 Gly Lys Glu Glu Pro Cys Phe Thr Glu Asp Gln Met Asp Glu Ala Leu 180 185 190 Lys Ala Ile Gly Tyr Lys Gly Pro Glu Pro Asp Leu Leu Leu Ile Tyr 195 200 205 Gly Pro Val Arg Cys His Leu Gly Phe Ser Pro Trp Arg Leu Arg Tyr 210 215 220 Thr Glu Met Val His Met Gly Pro Leu Arg Tyr Met Asn Leu Gly Ser 225 230 235 240 Leu Lys Lys Ala Ile His Arg Phe Thr Thr Val Gln Gln Asn Tyr Gly 245 250 255 Thr <210> 11 <211> 528 <212> DNA <213> Hevea brasiliensis <400> 11 atggctgaag gtgaagaaga ggtgaatatc caagaagagg caaataaagg agaggagaat 60 ccccaagaag aggcgaatat ccaagaagag acgaataaag gagaggagaa tatccaagaa 120 gaggcgaata tcgaagaaga ggctaataag gaggaagaga gcctaaagta tttggatttt 180 gtgcaagcgg ctacagttta tgccagggct tctttctcaa agctctacct ttttgccaag 240 gacaagtctg gtccattcaa gcctggcgtc aatactgttg agagtaggtt taagagcgtg 300 gttagacccg tctataataa gttccaacct gttcccaaca aggttctcaa gtttgcagac 360 cgtagggttg atgcatatgt cactgtttta gatcgcattg ttcctccaat tgtcaagcgg 420 gcatctatcc aagcttattc agtagcccca ggagctgctc gtgctgtggc ttcttatttg 480 cctttgcata ccaagagact ttttaaggtg ctatatggag atgggtga 528 <210> 12 <211> 175 <212> PRT <213> Hevea brasiliensis <400> 12 Met Ala Glu Gly Glu Glu Glu Val Asn Ile Gln Glu Glu Ala Asn Lys 1 5 10 15 Gly Glu Glu Asn Pro Gln Glu Glu Ala Asn Ile Gln Glu Glu Thr Asn 20 25 30 Lys Gly Glu Glu Asn Ile Gln Glu Glu Ala Asn Ile Glu Glu Glu Ala 35 40 45 Asn Lys Glu Glu Glu Ser Leu Lys Tyr Leu Asp Phe Val Gln Ala Ala 50 55 60 Thr Val Tyr Ala Arg Ala Ser Phe Ser Lys Leu Tyr Leu Phe Ala Lys 65 70 75 80 Asp Lys Ser Gly Pro Phe Lys Pro Gly Val Asn Thr Val Glu Ser Arg 85 90 95 Phe Lys Ser Val Val Arg Pro Val Tyr Asn Lys Phe Gln Pro Val Pro 100 105 110 Asn Lys Val Leu Lys Phe Ala Asp Arg Val Asp Ala Tyr Val Thr 115 120 125 Val Leu Asp Arg Ile Val Pro Ile Val Lys Arg Ala Ser Ile Gln 130 135 140 Wing Tyr Ser Val Wing Pro Gly Wing Wing Arg Wing Val Val Ser Tyr Leu 145 150 155 160 Pro Leu His Thr Lys Arg Leu Phe Lys Val Leu Tyr Gly Asp Gly 165 170 175 <210> 13 <211> 417 <212> DNA <213> Hevea brasiliensis (Hevea brasiliensis) <400> 13 atggctgaag acgaagacaa ccaacaaggg cagggggagg ggttaaaata ttttggttttt 60 gtgcaagacg cggcaactta tgctgtgact accttctcaa acgtctatct tttgccaaa 120 ɣaaatctg gtccactgca gcctggtgtc gatatcattg agggtccggt gagaacgtg 180 gctgtaccctc tctataatag gttcagttat attcccaatg gagctctcaa gtttgtagac 240 agcacggttg ttgcatctgt cactattata gatcgctctc ttcccccaat tgtcaaggac 300 gcatctatcc aagttgtttc agcaattcga gctgccccag aagctgctcg ttctctggct 360 tcttctttgc ctgggcagac caagatactt gctaaggtgt tttatggaga gaattga 417 <210> 14 <211> 138 <212> PRT <213> Hevea brasiliensis <400> 14 Met Ala Glu Asp Glu Asp Asn Gln Gln Gly Gln Gly Glu Gly Leu Lys 1 5 10 15 Tyr Leu Gly Phe Val Gln Asp Ala Ala Thr Tyr Ala Val Thr Thr Phe 20 25 30 Ser Asn Val Tyr Leu Phe Ala Lys Asp Lys Ser Gly Pro Leu Gln Pro 35 40 45 Gly Val Asp Ile Ile Glu Gly Pro Val Lys Asn Val Ala Val Pro Leu 50 55 60 Tyr Asn Arg Phe Ser Tyr Ile Pro Asn Gly Ala Leu Lys Phe Val Asp 65 70 75 80 Ser Thr Val Val Ala Ser Val Thr Ile Ile Asp Arg Ser Leu Pro Pro 85 90 95 Ile Val Lys Asp Ala Ser Ile Gln Val Val Ser Ala Ile Arg Ala Ala 100 105 110 Pro Glu Ala Ala Arg Ser Leu Ala Ser Ser Leu Pro Gly Gln Thr Lys 115 120 125 Ile Leu Ala Lys Val Phe Tyr Gly Glu Asn 130 135 <210> 15 <211> 354 <212> DNA <213> Hevea brasiliensis <400> 15 atggctgaag ggaaagaaaa cgagaatttc caacaagagg ctaatgagca ggaagagaag 60 ctaaagtatt tggaatttgt acaagcgact acagataatg ctgtaactgc cctctcaaac 120 atttaccttt atgccaagga caattctggt ccgttgaagc ctggggtcga gactattgag 180 ggtgtggcaa agaccgtggt tattccggcc agtaaaattc ccactgaagc tatcaagttt 240 gcagacagag cggtggatgc atctttcact actctacaaa acattgttcc ctcagttctc 300 aagcagttgc ctacccaagc ttgcgatact agtgtgaagg aaagtgcaga gtaa 354 <210> 16 <211> 117 <212> PRT <213> Hevea brasiliensis <400> 16 Met Ala Glu Gly Lys Glu Asn Glu Asn Phe Gln Gln Glu Ala Asn Glu 1 5 10 15 Gln Glu Glu Lys Leu Lys Tyr Leu Glu Phe Val Gln Ala Thr Thr Asp 20 25 30 Asn Ala Val Thr Ala Leu Ser Asn Ile Tyr Leu Tyr Ala Lys Asp Asn 35 40 45 Ser Gly Pro Leu Lys Pro Gly Val Glu Thr Ile Glu Gly Val Ala Lys 50 55 60 Thr Val Val Ile Pro Ala Ser Lys Ile Pro Thr Glu Ala Ile Lys Phe 65 70 75 80 Ala Asp Arg Ala Val Asp Ala Ser Phe Thr Thr Leu Gln Asn Ile Val 85 90 95 Pro Ser Val Leu Lys Gln Leu Pro Thr Gln Ala Cys Asp Thr Ser Val 100 105 110 Lys Glu Ser Ala Glu 115 <210> 17 <211> 615 <212> DNA <213> Hevea brasiliensis <400> 17 atggctgaag aggtggagga agagaggcta aagtatttgg attttgtgcg agcggctgga 60 gtttatgctg tagattcttt ctcaactctc tacctttatg ccaaggacat atctggtcca 120 ttaaaacctg gtgtcgatac tattgagaat gtggtgaaga ccgtggttac tcctgttat 180 tatattcccc ttgaggctgt caagtttgta gacaaaacgg tggatgtatc ggtcactagc 240 ctagatggcg ttgttccccc agttatcaag caggtgtctg cccaaactta ctcggtagct 300 caagatgctc caagaattgt tcttgatgtg gcttcttcag ttttcaacac tggtgtgcag 360 gaaggcgcaa aagctctgta cgctaatctt gaaccaaaag ctgagcaata tgcggtcatt 420 acctggcgtg ccctcaataa gctgccacta gttcctcaag tggcaaatgt agttgtgcca 480 accgctgttt atttctctga aaagtacaac gatgttgttc gtggcactac tgagcaggga 540 tatagagtgt cctcttattt gcctttgttg cccactgaga aaattactaa ggtgtttgga 600 gatgaggcat cataa 615 <210> 18 <211> 204 <212> PRT <213> Hevea brasiliensis <400> 18 Met Ala Glu Glu Val Glu Glu Glu Arg Leu Lys Tyr Leu Asp Phe Val 1 5 10 15 Arg Ala Ala Gly Val Tyr Ala Val Asp Ser Phe Ser Thr Leu Tyr Leu 20 25 30 Tyr Ala Lys Asp Ile Ser Gly Pro Leu Lys Pro Gly Val Asp Thr Ile 35 40 45 Glu Asn Val Val Lys Thr Val Val Thr Pro Val Tyr Tyr Ile Pro Leu 50 55 60 Glu Ala Val Lys Phe Val Asp Lys Thr Val Asp Val Ser Val Thr Ser 65 70 75 80 Leu Asp Gly Val Val Pro Pro Val Ile Lys Gln Val Ser Ala Gln Thr 85 90 95 Tyr Ser Val Ala Gln Asp Ala Pro Arg Ile Val Leu Asp Val Ala Ser 100 105 110 Ser Val Phe Asn Thr Gly Val Gln Glu Gly Ala Lys Ala Leu Tyr Ala 115 120 125 Asn Leu Glu Pro Lys Ala Glu Gln Tyr Ala Val Ile Thr Trp Arg Ala 130 135 140 Leu Asn Lys Leu Pro Leu Val Pro Gln Val Ala Asn Val Val Val Pro 145 150 155 160 Thr Ala Val Tyr Phe Ser Glu Lys Tyr Asn Asp Val Val Arg Gly Thr 165 170 175 Thr Glu Gln Gly Tyr Arg Val Ser Ser Tyr Leu Pro Leu Leu Pro Thr 180 185 190 Glu Lys Ile Thr Lys Val Phe Gly Asp Glu Ala Ser 195 200 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer 1, used to amplify CPT1 <400> 19 atggaattat acaacggtg 19 <210> 20 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Primer 2, used to amplify CPT1 <400> 20 atctcgagtt aatgatgatg atgatgatgt tttaag 36 <210> twenty one <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Primer 3 is used to amplify CPT2. <400> twenty one atggaaatat atacgggtca g 21 <210> twenty two <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Primer 4 is used to amplify CPT2. <400> twenty two atctcgagtt aatgatgatg atgatgatgt tttaaatatt c 41 <210> twenty three <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer 5 is used to amplify CPT6. <400> twenty three atggaaaaac atagcagtag 20 <210> twenty four <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer 6 is used to amplify CPT6. <400> twenty four atctcgagtt atataactga tgctttttc 29 <210> 25 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer 7 is used to amplify CPT7. <400> 25 atgcaatcct tgcacttg 18 <210> 26 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer 8 is used to amplify CPT7. <400> 26 atctcgagtt atgtaagtcg tctaccatag 30 <210> 27 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Primer 9 is used to amplify CPTL. <400> 27 atggatttga aacctgg 17 <210> 28 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Primer 10, used to amplify CPTL <400> 28 atctcgagtt aatgatgatg atgatgatgt gtac 34 <210> 29 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer 11 is used to amplify REF1. <400> 29 atggctgaag gtgaagaaga ggtgaatatc 30 <210> 30 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer 12 is used to amplify REF1. <400> 30 atctcgagtc acccatctcc atatagcac 29 <210> 31 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primer 13 is used to amplify REF2. <400> 31 atggctgaag acgaagacaa ccaacaag 28 <210> 32 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer 14 is used to amplify REF2. <400> 32 atctcgagtc aattctctcc ataaaacac 29 <210> 33 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer 15 is used to amplify REF8. <400> 33 atggctgaag ggaaagaaaa cgagaatttc 30 <210> 34 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer 16 is used to amplify REF8. <400> 34 atctcgagtt actctgcact ttccttcac 29 <210> 35 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primer 17 is used to amplify SRPP1. <400> 35 atggctgaag aggtggagga agagaggc 28 <210> 36 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer 18 is used to amplify SRPP1. <400> 36 atctcgagtt atgatgcctc atctccaaac 30

Claims

1. A lipid membrane construct, wherein the lipid membrane construct is a lipid bilayer membrane construct or lipoprotein body comprising a protein composition having isoprene polymerization activity, wherein the protein composition comprises: (A-1) CPT6 protein with amino acid sequence number 6, or (A-2) The CPT7 protein with sequence number 8. Furthermore, the protein composition comprises: (B) CPTL protein with amino acid sequence number 10.

2. The lipid membrane construct according to claim 1, wherein it satisfies at least one of the following: (A-1) is a protein encoded by polynucleotide (a-1), wherein polynucleotide (a-1) is a polynucleotide with the base sequence of sequence number 5. (A-2) is a protein encoded by polynucleotide (a-2), wherein polynucleotide (a-2) is a polynucleotide with the base sequence of sequence number 7, and (B) is a protein encoded by polynucleotide (b), which is a polynucleotide with the base sequence of sequence number 9.

3. The lipid membrane construct according to claim 1 or 2, wherein the composition further comprises CPT family proteins other than CPT6, 7 and CPTL, REF family proteins and SRPP family proteins.

4. The lipid membrane construct according to claim 1, wherein the composition further comprises at least one of the following: (C) The CPT1 protein with the amino acid sequence of sequence number 2. (D) The CPT2 protein with sequence number 4. (E) The REF1 protein with amino acid sequence number 12. (F) The REF2 protein with amino acid sequence number 14. (G) The REF8 protein with amino acid sequence number 16, and (H) SRPP1 protein with amino acid sequence number 18.

5. The lipid membrane construct according to claim 4, wherein it satisfies at least one of the following: (C) is the protein encoded by polynucleotide (c), wherein polynucleotide (c) is the polynucleotide with the base sequence of sequence number 1. (D) is the protein encoded by polynucleotide (d), wherein polynucleotide (d) is the polynucleotide with the base sequence of sequence number 3. (E) is the protein encoded by polynucleotide (e), wherein polynucleotide (e) is the polynucleotide with the base sequence of sequence number 11. (F) is the protein encoded by polynucleotide (f), wherein polynucleotide (f) is the polynucleotide with the base sequence of sequence number 13. (G) is a protein encoded by polynucleotide (g), wherein polynucleotide (g) is a polynucleotide with the base sequence of sequence number 15, and (H) is a protein encoded by a polynucleotide (h), which is a polynucleotide with the base sequence of sequence number 17.

6. The lipid membrane construct according to claim 5, wherein, At least one of the proteins (A-1), (A-2) and (B) to (H) is a protein derived from the rubber tree.

7. The lipid membrane construct according to claim 1 or 2, wherein the liposome further comprises phospholipids.

8. The lipid membrane construct of claim 7, wherein the phospholipid comprises phospholipids derived from soybean.

9. The lipid membrane construct according to claim 1 or 2, wherein the lipoprotein bodies are 50 to 300 nm in diameter.

10. A method for manufacturing a lipid membrane construct, wherein, The lipid membrane construct is the lipid membrane construct according to any one of claims 1 to 9. The method includes a step of expressing a polynucleotide in a cell-free protein production system in the presence of a phospholipid-containing raw material, wherein the polynucleotide encodes a protein constituting a composition comprising the construct of any one of claims 1 to 9.

11. The method for manufacturing the lipid membrane construct according to claim 10, wherein, The polynucleotide encoding the protein constituting the composition of any one of claims 1 to 9 comprises either (a-1) or (a-2), and comprises (b): (a-1) The polynucleotide sequence of sequence number 5, (a-2) The polynucleotide sequence of sequence number 7, (b) The polynucleotide sequence of sequence number 9.

12. The method for manufacturing a lipid membrane construct according to claim 10 or 11, wherein, The polynucleotide is a polynucleotide encoding a protein constituting the composition comprising the construct of claim 3 or 4, further comprising: (c) The polynucleotide sequence of sequence number 1, (d) The polynucleotide sequence of sequence number 3, (e) The polynucleotide sequence of sequence number 11, (f) The polynucleotide sequence of sequence number 13, (g) The polynucleotide sequence of sequence number 15, and (h) The polynucleotide sequence of sequence number 17.

13. A method for manufacturing an isoprene polymer compound, comprising a step of performing an isoprene polymerization reaction using the lipid membrane construct according to any one of claims 1 to 9.

14. The manufacturing method according to claim 13, wherein, Isoprene polymerization was carried out using low molecular weight allyl compounds as substrates.

15. A kit for manufacturing isoprene polymer compounds, comprising: The lipid membrane construct according to any one of claims 1 to 9, and the low molecular weight allyl compound.

16. A method for manufacturing rubber, comprising the steps of manufacturing an isoprene polymer compound by the manufacturing method of claim 13 or 14 and manufacturing rubber using the isoprene polymer compound.

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