CN102391999B - Homocysteine methyltransferase, nucleotide sequence thereof, recombinant vector, recombinant host cells, manufacturing method and kit - Google Patents
Homocysteine methyltransferase, nucleotide sequence thereof, recombinant vector, recombinant host cells, manufacturing method and kit Download PDFInfo
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- CN102391999B CN102391999B CN2011103436413A CN201110343641A CN102391999B CN 102391999 B CN102391999 B CN 102391999B CN 2011103436413 A CN2011103436413 A CN 2011103436413A CN 201110343641 A CN201110343641 A CN 201110343641A CN 102391999 B CN102391999 B CN 102391999B
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- homocysteine methyltransgerase
- methyltransgerase
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Abstract
The invention discloses a homocysteine methyltransferase, the amino acid sequence of the homocysteine methyltransferase is the amino acid sequence as shown in (1) SEQ ID NO: 1 (sequence identity number: 1), or the amino acid sequence as shown in (2) SEQ ID NO: 3 or the (3) amino acid sequence which is obtained by performing deletion, addition and/or substitution on one or a plurality of amino acids in the amino acid sequence described in (1) or (2), but has the constant functions of the homocysteine methyltransferase. The invention further discloses a nucleotide sequence for encoding the homocysteine methyltransferase, a recombinant vector including the nucleotide sequence, recombinant host cells including the recombinant vector, a method for purifying the enzyme from the recombinant host cells and a kit including at least one of the homocysteine methyltransferase, the nucleotide sequence, the recombinant vector and the recombinant host cells. The homocysteine methyltransferase is good in heat resistance and very high in stability.
Description
Technical field
The present invention relates to a kind of enzyme, the encode nucleotide sequence of this enzyme, the recombinant vectors that comprises described nucleotide sequence, the recombinant host cell that comprises described recombinant vectors, the method of this enzyme of purifying from aforementioned recombinant host cell, and comprise at least a test kit in above-mentioned enzyme, nucleotide sequence, recombinant vectors and the recombinant host cell.More specifically, the present invention relates to a kind of homocysteine methyltransgerase, the nucleotide sequence of this homocysteine methyltransgerase of encoding, the recombinant vectors that comprises described nucleotide sequence, the recombinant host cell that comprises described recombinant vectors, the method of this enzyme of purifying from aforementioned recombinant host cell, and comprise at least a test kit in above-mentioned homocysteine methyltransgerase, nucleotide sequence, recombinant vectors and the recombinant host cell.
Background technology
Methyltransgerase is the enzyme that plays crucial regulating effect in the vital movement, and it can be with the Methyl transporters on the S-adenosylmethionine (SAM) to the substrate molecule, generates the multiple important methyl physiologically active substance that contains, and reaches the purpose that function is regulated.Homocysteine methyltransgerase (EC 2.1.1.10) is a kind of in the methyltransgerase, can be used for the preparation of homocysteine (HCY) biochemical diagnosis reagent, is a kind of critical materials enzyme.
Existing homocysteine methyltransgerase is mainly extracted from yeast saccharomyces cerevisiae, exists the enzyme productive rate low, the shortcomings such as purification difficult, poor heat stability (it is complete deactivation that storage temperature surpasses 40 ℃).
Summary of the invention
An object of the present invention is to overcome the shortcoming of existing homocysteine methyltransgerase poor heat resistance, the low purification difficult of productive rate, the homocysteine methyltransgerase of a kind of good heat resistance, the high and easy purifying of productive rate is provided.
Second purpose of the present invention provides the nucleotide sequence of the described homocysteine methyltransgerase of coding.
The 3rd purpose of the present invention provides the recombinant vectors that comprises described nucleotide sequence.
The 4th purpose of the present invention provides the recombinant host cell that comprises described recombinant vectors.
The 5th purpose of the present invention provides at least a test kit that comprises in above-mentioned homocysteine methyltransgerase, nucleotide sequence, recombinant vectors and the recombinant host cell.
The present inventor has paid in large quantities creative work, from Candida albicans (Candida albicans SC5314), obtain the homocysteine methyltransgerase that the present invention has the aminoacid sequence shown in the SEQ ID NO:1, and utilize the method for random mutation to obtain the homocysteine methyltransgerase that the present invention has the aminoacid sequence shown in the SEQ ID NO:3.And the present inventor is surprised to find that two kinds of homocysteine methyltransgerase of gained all have good thermotolerance, can keep higher stability in wide temperature range.
The invention provides a kind of homocysteine methyltransgerase, wherein, the aminoacid sequence of described homocysteine methyltransgerase is
(1) aminoacid sequence shown in the SEQ ID NO:1, perhaps
(2) aminoacid sequence shown in the SEQ ID NO:3, perhaps
(3) (1) or (2) described aminoacid sequence is carried out one or several amino acid whose disappearance, interpolation and/or replacement, but the constant aminoacid sequence of the function of its homocysteine methyltransgerase.
The present invention also provides the nucleotide sequence of the homocysteine methyltransgerase of the present invention of encoding.
The present invention also provides the recombinant vectors that comprises nucleotide sequence of the present invention.
The present invention also provides the recombinant host cell that comprises recombinant vectors of the present invention.
The present invention also provides the method for this enzyme of purifying from aforementioned recombinant host cell.
The present invention also provides at least a test kit that comprises in homocysteine methyltransgerase of the present invention, nucleotide sequence, recombinant vectors and the recombinant host cell.
Homocysteine methyltransgerase provided by the invention has the advantage of good heat resistance.Referring to Fig. 3 as can be known, two kinds of homocysteine methyltransgerase of the present invention still all have the relative reactivity more than 60% after leaving standstill 15 minutes under 40 ℃ in the damping fluid of pH6.0, wherein, the homocysteine methyltransgerase of the present invention with the aminoacid sequence shown in the SEQ ID NO:3 still all has the relative reactivity more than 50% after leaving standstill 15 minutes under 45 ℃ in the damping fluid of pH6.0.
Description of drawings
The isogenic sepharose of the homocysteine methyltransgerase of Fig. 1 embodiment 1 is identified photo;
The SDS-PAGE of the homocysteine methyltransgerase of Fig. 2 embodiment 1 expression and purification after IPTG induces identifies photo, wherein, the M swimming lane is molecular weight marker, the protein expression of the 1st swimming lane for not induced by IPTG, the 2nd swimming lane is the protein expression after IPTG induces, can find out obviously that therefrom in the 2nd swimming lane, the target protein homocysteine methyltransgerase has obtained great expression;
Fig. 3 embodiment 1 and 2 homocysteine methyltransgerase thermostability figure.
Embodiment
The invention provides a kind of homocysteine methyltransgerase, wherein, the aminoacid sequence of described homocysteine methyltransgerase is
(1) aminoacid sequence shown in the SEQ ID NO:1, perhaps
(2) aminoacid sequence shown in the SEQ ID NO:3, perhaps
(3) (1) or (2) described aminoacid sequence is carried out one or several amino acid whose disappearance, interpolation and/or replacement, but the constant aminoacid sequence of the function of its homocysteine methyltransgerase.
Those skilled in the art are known, and 20 seed amino acid residues of constitutive protein matter can be divided into four classes according to side chain polarity:
1, nonpolar amino acid: L-Ala (Ala), α-amino-isovaleric acid (Val), leucine (Leu), Isoleucine (Ile), methionine(Met) (Met), phenylalanine (Phe), tryptophane (Trp) and proline(Pro) (Pro);
2, the uncharged amino acid of polarity: glycine (Gly), Serine (Ser), Threonine (Thr), halfcystine (Cys), l-asparagine (Asn), glutamine (Gln) and tyrosine (Tyr);
3, positively charged amino acid: arginine (Arg), Methionin (Lys) and Histidine (His);
4, electronegative amino acid: aspartic acid (Asp) and L-glutamic acid (Glu) (referring to " biological chemistry " (second edition) first volume, Shen is same, Wang Jingyan, 82-83 page or leaf, Higher Education Publishing House, December nineteen ninety).If belonging to the amino-acid residue of a classification in the protein together replaces, for example replace Lys or replace Ile by Leu by Arg, described residue role (such as positive charge being provided or forming the effect of hydrophobic pouch structure) in protein domain does not change, therefore can't exert an influence to the three-dimensional arrangement of protein, therefore still can realize the function of albumen.For example, as well known to those skilled in the art, Ala and Ser, Val and Ile, Asp and Glu, Ser and Thr, Ala and Gly, Ala and Thr, Ser and Asn, Ala and Val, Ser and Gly, Tyr and Phe, Ala and Pro, Lys and Arg, Asp and Asn, Leu and Ile, Leu and Val, Ala and Glu and Asp and Gly, mutually replace between any two, can not affect three-dimensional arrangement and the function of albumen.The described amino-acid residue that belongs to a classification together replaces on any one amino acid residue position that can occur on the homocysteine methyltransgerase.On the contrary, different classes of amino-acid residue replaces, and perhaps amino acid whose replacement does not meet the above-mentioned replacement rule of enumerating, and the structure of albumen is changed, and difference appears in function.
Homocysteine methyltransgerase provided by the invention can also be modified or suddenly change, the protein that obtains deriving." protein of deriving " of the present invention refers to have difference on the aminoacid sequence with the homocysteine methyltransgerase with above-mentioned aminoacid sequence, and the difference on the modified forms that does not affect sequence is perhaps arranged, and perhaps haves both at the same time.These albumen comprise genetic variant natural or that induce.Described induce variation body can obtain by various technology, such as the random mutation that radiation or mutagenic compound etc. produce, and also can be by obtaining such as fixed-point mutation method or the biological technology of other known moleculars.Described " protein of deriving " also comprises the analogue (such as D type amino acid) with the amino acid whose residue of natural L-type, and the analogue with that non-natural exists or synthetic amino acid (such as beta-amino acids, gamma-amino acid etc.).
(usually the not changing primary structure) form of modification comprises: the chemically derived form of the albumen that body is interior or external, and such as acetylize or carboxylated.Modification also comprises glycosylation, carries out glycosylation modified and albumen that produce in the procedure of processing such as those in the synthetic and processing of albumen or further.This modification can be carried out glycosylated enzyme (such as mammiferous glycosylase or deglycosylating enzyme) and finishes by albumen is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (such as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the albumen that has been improved its anti-proteolysis performance or optimized solubility property by modifying.
Under the preferable case, the aminoacid sequence of described homocysteine methyltransgerase is the aminoacid sequence shown in SEQ ID NO:1 or the SEQ ID NO:3.
The present invention also provides the nucleotide sequence of the homocysteine methyltransgerase of the present invention of encoding.
Known in this field, in 20 kinds of different amino acid of constitutive protein matter, except Met (ATG) or Trp (TGG) were respectively single password coding, other 18 seed amino acids were respectively by 2-6 codon encode (Sambrook etc., molecular cloning, press of cold spring harbor laboratory, New York, the U.S., second edition, 1989, see 950 pages of appendix D).Namely because the degeneracy of genetic codon, determine more than one mostly of an amino acid whose codon, the displacement of the 3rd Nucleotide often can not change amino acid whose composition in the triplet codon, and the nucleotide sequence of the gene of the same protein of therefore encoding can be different.Those skilled in the art are according to known password sublist, from aminoacid sequence disclosed by the invention, and the active constant aminoacid sequence of the homocysteine methyltransgerase that is obtained by described aminoacid sequence, can derive their nucleotide sequence of gene of to encode fully, nucleotide sequence as described in obtaining by biological method (such as PCR method, mutation method) or chemical synthesis process, so this partial nucleotide sequence all should be included in the scope of the present invention.On the contrary, utilize dna sequence dna disclosed herein, also can be by means commonly known in the art, such as method (molecular cloning, the press of cold spring harbor laboratory of Sambrook etc., New York, the U.S., second edition, 1989) carry out, by revising nucleotide sequence provided by the invention, obtain and the active consistent aminoacid sequence of homocysteine methyltransgerase of the present invention.
Under the preferable case, nucleotides sequence of the present invention is classified as
(1) nucleotide sequence shown in the SEQ ID NO:2; Perhaps
(2) nucleotide sequence shown in the SEQ ID NO:4; Perhaps
(3) nucleotide sequence shown in SEQ ID NO:2 or the SEQ ID NO:4 is carried out the nucleotide sequence that one or several Nucleotide replaces, lacks or increase and obtains, this nucleotide sequence coded homocysteine methyltransgerase function is constant.
Nucleotide sequence provided by the invention can obtain with the method for pcr amplification method, recombination method or synthetic usually.For example, those skilled in the art can be easy to obtain template and primer according to nucleotide sequence provided by the present invention and recombinant bacterial strain, utilize PCR to increase and obtain relevant sequence.When sequence is longer, can carry out twice or pcr amplification repeatedly, then the gained fragment be pressed the proper order splicing.In case obtained relevant nucleotide sequence, just can use the relevant aminoacid sequence of the large batch of acquisition of recombination method.Usually the gained nucleotide sequence is cloned into carrier, again in the transgene engineering bacteria, then the host cell of the method by routine after the propagation separates and obtains relevant nucleotide sequence.In addition, also can synthesize relevant nucleotide sequence with the synthetic method of known artificial chemistry.
The present invention also provides the recombinant vectors that comprises nucleotide sequence of the present invention.Known in this field, described recombinant vectors generally comprises empty carrier and inserts the goal gene of this empty carrier, and described goal gene is the nucleotide sequence of homocysteine methyltransgerase of the present invention.
In the present invention, various carrier known in the art can be selected in described " empty carrier " (or title " carrier "), such as commercially available various plasmids, clay, phage and retrovirus etc., the carrier that the preferred described empty carrier of the present invention is the lac promotor, more preferably described empty carrier is selected from the vehicle group that is comprised of pET serial carrier (for example pET30a), pUC19, pGEM and pBluescript.Described empty carrier can comprise multiple certification mark commonly used (reporter genes such as fluorescent mark, antibiotic marker) and restriction enzyme site.Construction of recombinant vector can adopt the various endonucleases (as can be with NcoI and XhoI etc. for pET30a) of the multiple clone site of empty carrier own to carry out enzyme and cut the acquisition linear plasmid, be connected with the gene fragment that adopts the cutting of identical nucleic acid restriction endonuclease, obtain recombinant plasmid.
The present invention also provides the recombinant host cell that comprises recombinant vectors of the present invention.
Can described recombinant vectors be transformed, transduce or be transfected in the host cell by the method for this area routine, such as Calcium Chloride Method chemical conversion, electroporation, preferred electric shock transforms; Described host cell can be prokaryotic cell prokaryocyte or eukaryotic cell, be preferably intestinal bacteria, Bacillus subtilus, yeast (such as pichia spp) or various animal and plant cells, more preferably described host cell is this area genetic engineering bacterium commonly used, such as intestinal bacteria, subtilis or pichia spp.Further preferred described host cell is e. coli bl21 (DE3), BL21, TOP10 or DH5a.Most preferably described host cell is e. coli bl21 (DE3).The substratum of described host cell and culture condition can adopt substratum and the culture condition of this area routine.And colibacillary medium optimization comprises: the potassium primary phosphate of 0.03-0.3% (or 0.03-1.5% SODIUM PHOSPHATE, MONOBASIC), the Sodium phosphate dibasic of 0.1-1% (or dipotassium hydrogen phosphate), the ammonium chloride of 0.05-0.5% (or sodium-chlor of 0.05-1%), the sal epsom of 0.01-0.2%, the yeast powder of 0.5-5% (yeast extract), the peptone of 0.1-1% (for example Tryptones), the glucose of 0.001-2%, the glycerine of 2.5%-10%, the lactose of 0.1%-0.8%, the oleic acid of 0.1-2%, the Yelkin TTS of 0.1-1%, and regulate pH to 6.8～7.2.Preferably, when cultivating described host cell and be e. coli bl21 (DE3), in initial medium, access the LB seed of the OD600=2 of 5%～20% volume～4, in 37 ℃, dissolved oxygen uses ammoniacal liquor, sulfuric acid to regulate pH6.5～pH7.3 greater than self-sow under 20% the condition.(when dissolved oxygen DO value rises to 40%) is cooled to 32 ℃ when nutritive ingredient exhausts in the initial substratum, ratio adding feed supplement liquid 1 according to 60～100ml/L begins to induce, and add feed supplement liquid 2 according to the speed constant speed of 5～10ml/h/L, use ammoniacal liquor to regulate pH.At induction period, reduced by 1 ± 0.1 ℃ every 3 ± 1 hours, be down to 28 ± 0.5 ℃ after constant temperature to fermentation ends.Wherein, the culture medium prescription of using among the present invention is as follows successively:
Seed culture medium:
Tryptones 10g/L, yeast extract 5g/L, sodium-chlor 10g/L.Sterilized 20 minutes for 121 ℃, add sulphuric acid kanamycin to final concentration 50mg/L after the sterilization cooling.
The fermentation initial medium:
Tryptones 10g/L, yeast extract 5g/L, dipotassium hydrogen phosphate 6～12g/L, SODIUM PHOSPHATE, MONOBASIC 3～6g/L, Citric acid monohydrate Food grade 0.2～1g/L, sal epsom 0.2～2g/L, micro-mother liquor 1～10ml/L, transfer pH to 6.8～7.2,121 ℃ sterilization 20 minutes.The glucose that adds sterilization before the inoculation uses ammoniacal liquor to transfer pH to 6.5～7.3 to final concentration 15～20g/L.Add sulphuric acid kanamycin to final concentration 50mg/L.
Phosphatic ratio is in initial medium: dipotassium hydrogen phosphate: SODIUM PHOSPHATE, MONOBASIC=2: 1
The prescription of trace element is:
10g FeSO
4.7H
2O, 2.25g ZnSO
4.7H
2O, 1g CuSO
4.5H
2O, 0.5gMnSO4.5H
2O, 0.23g Na
2B
4O
7.10H
2O, 2g CaCl
2.2H
2O, 0.1g (NH
4)
6Mo
7O
24, be dissolved in the 5M hydrochloric acid.
Feed supplement liquid 1:
Yeast extract 250 ± 50g/L, Tryptones 100 ± 20g/L, lactose 10 ± 2g/L, 121 ℃ of sterilization 20min.
Feed supplement liquid 2:
Glycerine 400 ± 50g/L, 121 ℃ of sterilization 20min.
Can use this area method commonly used from recombinant host cell, to separate and the purifying homocysteine methyltransgerase.For example, centrifugation substratum and recombinant host cell, cell debris, affinitive layer purification homocysteine methyltransgerase are removed in high-pressure homogenization smudge cells, centrifuging.For the product of the homocysteine methyltransgerase of separation and purification gained, can use this area method commonly used to carry out Purity.For example, Xylene Brilliant Cyanine G method, Kjeldahl determination, biuret method, lowry method, ultraviolet absorption method, affinity chromatography, enzymic activity, antigen-antibody method, electrophoretic analysis (such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis), analysis by sedimentation, diffusion analysis, permanent solubility method, protein spectrum etc.When smudge cells, generally all can give birth to heat, usually need in smudge cells, lower the temperature, need heat radiation after the smudge cells process is finished, then just can carry out next step purifying work; Because homocysteine methyltransgerase good heat resistance of the present invention, the smudge cells process need not temperature-fall period, even if the temperature of smudge cells liquid can reach 40-45 ℃ behind the smudge cells, need not heat radiation, still can directly carry out next step purification step at once.For example the enchylema after the fragmentation adds calcium chloride, centrifugal (, remove most of membranin and nucleic acid; In centrifugal gained supernatant, add again the nickel ion protein precipitation.The albumen of gained precipitation can adopt following prescription to redissolve: the ethylenediamine tetraacetic acid (EDTA) (EDTA) of 10～50mmol/L pH7.0～8.0 phosphate buffered saline buffers, 2～20mmol/L Reduced nicotinamide-adenine dinucleotide (NAD) and 5～10mmol/L.Because therefore homocysteine methyltransgerase good heat resistance of the present invention can also adopt the broken liquid of reconstitution cell was heated 15-40 minute at 40-45 ℃, then at the centrifugal 10-20min of 8000-15000rpm, remove heat labile foreign protein in addition.Preferably, adopt the broken liquid of reconstitution cell is heated 30min at 45 ℃, the centrifugal 15min of 10000rpm removes most of heat labile foreign protein.More preferably, according to the following step purifying homocysteine methyltransgerase:
(1) fermented liquid is transferred pH to 7.0～8.0,600～900bar homogenate once, and the target protein homocysteine methyltransgerase is discharged.
(2) add the calcium chloride of 30～100mM in the homogenate, abundant stirring and evenly mixing, centrifugal 20 minutes of 5000g removes most nucleic acid, cell debris, and the part foreign protein.
(3) add 1～5mM nickel ion (nickel ions of various salt forms) in centrifugal supernatant, mixing left standstill under the room temperature 1 hour, the centrifugal 20min of 5000g, and most target proteins are in precipitation.
(4) the redissolution damping fluid that adds 1/5～1 times of volume of original volume in the precipitation stirred about 30 minutes.Centrifugal 20 minutes of 10000g.The buffer formulation of redissolving is 10～50mM pH7.0～8.0 phosphate buffered saline buffers (PB), 2～20mM NAD, 5～10mM EDTA.
(5) redissolution supernatant desalination behind 0.45 μ m membrane filtration.The desalination buffer formulation is 10～50mMpH7.0～8.0PB, 2～5mM EDTA.
The present invention also provides at least a test kit that comprises in homocysteine methyltransgerase of the present invention, nucleotide sequence, recombinant vectors and the recombinant host cell.Under the preferable case, test kit of the present invention comprises homocysteine methyltransgerase, and do not comprise nucleotide sequence, recombinant vectors and recombinant host cell.More preferably test kit of the present invention comprises the dry powder of homocysteine methyltransgerase, and does not comprise nucleotide sequence, recombinant vectors and recombinant host cell.The dry powder that can prepare with this area method commonly used homocysteine methyltransgerase, as long as the method can access the dry powder of homocysteine methyltransgerase, and the activity of not destroying the dry powder of homocysteine methyltransgerase gets final product.Then the enzyme liquid that for example uses the vacuum-concentrcted device to obtain concentrating use the dry concentrated enzyme liquid of freeze drier; Perhaps use the equipment such as vacuum decompression moisture eliminator.Be converted into liquid form in the buffer solution system (for example acetate buffer, phosphate buffered saline buffer, preferred pH is 5.5-7.0) that the dry powder of homocysteine methyltransgerase in use can be by being dissolved in pH5.0-8.0.
Test kit of the present invention can also comprise that this area is usually used in measuring the composition of the test kit of homocysteine.Can be liquid form or solid dry powder form corresponding to the homocysteine methyltransgerase in the test kit of the present invention, other compositions in the test kit of the present invention also can be liquid form and/or solid dry powder form.For example, when test kit of the present invention is liquid form, can contain 0.01-0.5mol/L pH 8.0Tris-Cl salt buffer in the reaction solution, the homocysteine methyltransgerase of 1000-10000U/L, the SAHH of 100-10000U/L, the adenosine deaminase of 100-10000U/L, the S-adenosylmethionine of 1-100mmol/L (SAM), the a-ketoglutaric acid of 1-20mmol/L, the glutamate dehydrogenase of 1000-100000U/L, 0.2-2mmol/L NADH (NADH) and the TCEP (TCEP) of 1-20mmol/L.Test kit of the present invention preferably contains 0.01-0.05mol/L pH 8.0 Tris-Cl salt buffers, the homocysteine methyltransgerase of 1000-10000U/L, the SAHH of 100-1000U/L, the adenosine deaminase of 1000-10000U/L, the S-adenosylmethionine of 1-15mmol/L (SAM), 1.2-10mmol/L a-ketoglutaric acid, the glutamate dehydrogenase of 1000-10000U/L, 0.3-1.5mmol/L NADH and the TCEP (TCEP) of 1-10mmol/L.The kit components of liquid form of the present invention can be passed through Freeze Drying Technique, in conjunction with reduced pressure distillation technique, reverse osmosis technology and ultra-filtration technique etc., is prepared into reagent dry powder.Described dry powder can redissolve to original volume of described reagent with the solvent that is selected from deionized water, distilled water and distilled water before detecting sample to be measured.Test kit of the present invention can comprise the specification sheets that records above-mentioned various component using method and consumption.Therefore, described solution also can be prepared according to prior art according to the record of test kit specification sheets before use by the user and get final product.
Being suitable for using the sample to be measured of test kit of the present invention can be under animal body (the comprising human body) state of health and the serum of separating out after the blood natural coagulation under the pathological state.
Further specify the present invention below in conjunction with embodiment, the used reagent of the present invention, substratum are the commercial goods unless stated otherwise.
Preparation Example 1
A) acquisition of natural homocysteine Methyl transporters gene (namely obtains the Nucleotide shown in the sequence 1
Sequence)
A-1) cultivation of Candida albicans (Candida albicans SC5314) (available from ATCC MYA-2876)
The solid medium that Candida albicans is cultivated is: 2% glucose, 1% peptone, 0.5% yeast extract paste and 2% agar, 121 ℃ of 20min sterilizations (" % " refers to weight percent if no special instructions, herein).The liquid nutrient medium that Candida albicans is cultivated is: 2% glucose, 1% peptone and 0.5% yeast extract paste, 121 ℃ of 20min sterilizations.
Candida albicans SC5314 activates at the above solid plate, and reactivation process was namely cultivated in 30 ℃ of incubators 36 hours, then with colony inoculation in above yeast liquid nutrient medium; Cultivated 24 hours under 30 ℃ of conditions.
A-2) extraction of candida albicans gene group DNA
Get step 1) culture that obtains, the centrifugal 5min of 10000rpm at room temperature, abandon supernatant, precipitation Eddy diffusion (2% (v/v) Triton X-100,1% (m/v) SDS, 100mM NaCl in 400 μ l lysis buffers, 10mM Tris-Cl (pH8.0), 1mM EDTA (pH8.0)), add the pickling glass pearl that equates with cell volume, and 400 μ l phenol/chloroforms.Ice bath, high vibration 5 * 30s.The centrifugal 10min of 10000rpm transfers to supernatant in the new EP pipe.Add 0.6 times of volume Virahol or 2 times of volume ethanol, put upside down mixing, room temperature is placed 10min.The centrifugal 10min of 10000rpm abandons supernatant.Add 500 μ l, 75% ethanol, vortex, the centrifugal 2min of 10000rpm abandons supernatant.To be deposited in air drying 10min, add 50 μ l TE (10mM Tris-Cl (pH8.0), 1mM EDTA (pH8.0)) dissolving.The RNase that adds the 10mg/ml of 1 μ l, 37 ℃ of lower digestion 30min.Obtain genomic dna.
A-3) amplification homocysteine methyltransgerase gene
The homocysteine methyltransgerase gene primer of inventor's design is as follows:
Homocysteine methyltransgerase gene forward primer (SAMF) (SEQ ID NO:5):
ATCGCCATGGGACGTGTTCAGGATATTTTAG; Near the NcoI restriction enzyme site is arranged.
Homocysteine methyltransgerase gene reverse primer (SAMR) (SEQ ID NO:6):
ATCGCTCGAGCTATTGGTTGATCAATTGTGCAAC; Near the XhoI restriction enzyme site is arranged.
Amplification condition is: 20mM Tris-HCl (pH 8.8), 10mM KCl, 10mM (NH
4)
2SO
4, 2mM MgSO
4, 0.1%Triton X-100,50 μ M dATP, 50 μ M dGTP, 50 μ M dTTP, 50 μ M dCTP, 400nM primer SAMF, 400nM primer SAMR, 1.5U Pfu archaeal dna polymerase (Promega, USA), the 20ng genomic dna adds reaction system, transfers to reaction volume with sterilized water and reaches 50 μ L.
The amplified reaction program is: above-mentioned reaction system 95 ℃ of lower reactions 5 minutes, is then carried out " 95 ℃ 30 seconds, 55 ℃ 30 seconds and 72 ℃ 2 minutes " of 30 circulations, at last 72 ℃ of lower maintenances 10 minutes.The pcr amplification gene product that obtains is separated with 1% agarose gel electrophoresis, and reclaim the dna fragmentation of the single band in the 936bp left and right sides with QIAGEN rapid extraction gel reagents box.
B) structure of recombinant vectors
With steps A) the homocysteine methyltransgerase gene that obtains is through NcoI and XhoI double digestion 4h, with same pET30a carrier through NcoI and XhoI double digestion 4h, by using the T4DNA ligase enzyme to connect the two, be built into the homocysteine methyltransgerase recombinant expression vector.
C) recombinant vectors is to the conversion of host cell and the cultivation of gained recombinant host cell
Picking is 37 ℃ of single bacterium colonies of cultivating 16 hours e. coli bl21 (DE3) on the LB solid medium, cultivate 16 hours at 37 ℃, the shaking table of 150rpm in liquid LB substratum again.Get 1ml gained liquid culture and transfer in 100ml fresh liquid LB substratum, the rotating speed thermal agitation with 250-300rpm on 37 ℃ of shaking tables was cultivated 2.5 hours.Draw the cultured bacterium liquid of 1.5ml to the 1.5ml centrifuge tube, in cooled on ice after 10 minutes, under 4 ℃, 3000g centrifugal 5 minutes.Supernatant discarded adds 100 μ l at the CaCl2 of the 0.1mol/L of precooling on ice solution, inhales up and down gently moving beating with liquid-transfering gun and spares, and re-suspended cell was placed 20 minutes at ice.Then under 4 ℃, 3000g centrifugal 5 minutes, supernatant discarded added 100 μ l at the CaCl of the 0.1mol/L of precooling on ice
2Solution is inhaled up and down gently moving beating with liquid-transfering gun and is spared, and re-suspended cell obtains competent host cell.
Get the above-mentioned competent e. coli bl21s of 200 μ l (DE3) and place the 1.5ml centrifuge tube, the adding volume is that the pET30a solution (wherein pET30a content is 40ng) of 10 μ l shakes up gently, places on ice 30 minutes.Then thermal shock 90 seconds in 42 ℃ of water-baths then placed rapidly cooled on ice 5 minutes.Add 1ml LB liquid nutrient medium (not containing kantlex) in the centrifuge tube, 37 ℃ of shaking culture are 1 hour behind the mixing, make bacterium restore normal growth state and the kantlex antibiotics resistance gene of expression plasmid coding.Get 100 μ l after above-mentioned bacterium liquid shaken up and coat on the LB screening flat board that contains Kan, face up and place half an hour, after bacterium liquid is absorbed by substratum fully, be inverted culture dish, cultivated 20 hours for 37 ℃.Colibacillary bacterium colony occurred at the screening flat board, namely pET30a has been converted in e. coli bl21 (DE3) competent cell, has obtained the recombinant host cell of present embodiment---e. coli bl21 (DE3) pET30a.
D) cultivation of recombinant host cell
With above-mentioned steps C) the corresponding e. coli bl21 (DE3) with the nucleotide sequence of coding homocysteine methyltransgerase that obtains is cloned in and is cultured to growth logarithmic phase (OD600=2) in the LB substratum under 37 ℃, be inoculated in the 3L initial medium, namely in initial medium, access the LB seed liquor of the OD600=2 of 10% volume, in 37 ℃, dissolved oxygen uses ammoniacal liquor, sulfuric acid to regulate pH6.5～pH7.3 greater than self-sow under 20% the condition.(when dissolved oxygen DO value rises to 40%) is cooled to 32 ℃ when nutritive ingredient exhausts in the initial substratum, ratio adding feed supplement liquid 1 according to 60～100ml/L begins to induce, and add feed supplement liquid 2 according to the speed constant speed of 5～10ml/h/L, use ammoniacal liquor to regulate pH.At induction period, reduced by 1 ± 0.1 ℃ every 3 ± 1 hours, be down to after 28 ± 0.5 ℃ constant temperature to fermentation ends (be the enzyme work of homocysteine methyltransgerase in the fermented liquid greater than 200KU/L, the enzyme of wherein said homocysteine methyltransgerase is lived and measured with reference to the enzyme activity determination method of accompanying homocysteine methyltransgerase).Wherein, the culture medium prescription of using among the present invention is as follows successively:
Seed culture medium:
Tryptones 10g/L, yeast extract 5g/L, sodium-chlor 10g/L.Sterilized 20 minutes for 121 ℃, add sulphuric acid kanamycin to final concentration 50mg/L after the sterilization cooling.
The fermentation initial medium:
Tryptones 10g/L, yeast extract 5g/L, dipotassium hydrogen phosphate 8g/L, SODIUM PHOSPHATE, MONOBASIC 4g/L, Citric acid monohydrate Food grade 0.5g/L, sal epsom 1g/L, micro-mother liquor 5ml/L transfer pH to 7.0, sterilize 20 minutes for 121 ℃.The glucose that adds sterilization before the inoculation uses ammoniacal liquor to transfer pH to 7.0 to final concentration 18g/L.Add sulphuric acid kanamycin to final concentration 50mg/L.
Phosphatic ratio is in initial medium: dipotassium hydrogen phosphate: SODIUM PHOSPHATE, MONOBASIC=2: 1
The prescription of trace element is:
10g FeSO
4.7H
2O, 2.25g ZnSO
4.7H
2O, 1g CuSO
4.5H
2O, 0.5gMnSO
4.5H
2O, 0.23g Na
2B
4O
7.10H
2O, 2g CaCl
2.2H
2O and 0.1g (NH
4)
6Mo
7O
24, be dissolved in the 5M hydrochloric acid.
Feed supplement liquid 1:
Yeast extract 250g/L, Tryptones 100g/L, lactose 10g/L, 121 ℃ of sterilization 20min.
Feed supplement liquid 2:
Glycerine 400g/L, 121 ℃ of sterilization 20min.
E) separating-purifying of protein product
With step D) fermented liquid that obtains adjusts pH to 7.0-8.0, with the resuspended cell of 800bar high-pressure homogenization crusher machine, first with smudge cells liquid at 45 ℃ of lower heating 30min, target protein is discharged.The calcium chloride that adds 50mM in the homogenate, abundant stirring and evenly mixing, centrifugal 20 minutes of 5000g removes most nucleic acid, cell debris, and the part foreign protein.Add 4mM nickel ion (single nickel salt) in centrifugal supernatant, mixing left standstill under the room temperature 1 hour, the centrifugal 20min of 5000g, and most target proteins are in precipitation.The redissolution damping fluid that adds 1 times of volume of original volume in the precipitation stirred about 30 minutes.Centrifugal 20 minutes of 10000g.The liquid formula that redissolves is 40mM pH7.5PB, 10mM NAD, 8mM EDTA.Redissolve supernatant through 0.45 μ m membrane filtration.
Gained filtrate is used sephadex G-25 chromatography column (Sephadex G-25) desalination again.Described desalination buffer formulation is the PB of 40mM pH7.5,4mM EDTA.Use loading on the good chromatography column of desalination damping fluid balance, wherein the flow velocity of sample solution is 5ml/min again, then uses the desalination damping fluid with 2 column volumes of flow velocity wash-out of 15ml/min.Totally 500 milliliters of liquid under the collection wash-out.
Collected liquid in general refrigerator-10 ℃ lower freezing 2 hours, and then-40 ℃ of cryogenic refrigerator pre-freezes 8 hours, in the ALPHA 1-4LSC of German Ke Ruisite (CHRIST) type freeze drier, with the condition freeze-drying of vacuum tightness 0.04mbar, safe pressure 0.100mbar and condenser temperature-60 ℃ 25 hours.Then waiting temperature of charge and Freeze Drying Equipment baffle temperature poor is zero, and observes 15 seconds internal pressures and indicate when constant, finishes freeze-drying.The amount of freeze-drying gained protein is 12 grams.Lyophilized protein under freezing conditions seals preservation.The freeze-drying sample that takes a morsel records enzyme with reference to the enzyme activity determination method of accompanying homocysteine methyltransgerase and lives and be 100U/1mg.
Adopt " protein electrophorese experimental technique " (Guo Yaojun, the 132-145 page or leaf, Science Press, 1999) method of SDS-PAGE electrophoresis of the record molecular weight that determines the protein of present embodiment is 36KD, and according to " biological chemistry " (Wang Jingyan etc., Higher Education Publishing House, 2002, see 168 pages) measuring method (minute peptide section measure) of the prlmary structure of protein of record, the protein that records present embodiment has 330 amino-acid residues (referring to the aminoacid sequence shown in the SEQ ID NO:1), with consistent by the nucleotide sequence coded protein result shown in the SEQ ID NO:2.
Measure in accordance with the following methods the activity of homocysteine methyltransgerase:
Reagent one: the TRIS of weighing 3g adds the ZnSO of 0.1435g with the deionized water dissolving of 400ml
4.7H
2Then O adjusts PH to 7.5 with hydrochloric acid, and contain 1mM zine ion for 50mM Tris-HCl this moment, adds 100mM KCl in this solution.
Reaction reagent: in the solution of reagent one, add respectively 5mM homocysteine (HCY) and 5mM S-adenosylmethionine (SAM) (now with the current).
Reaction principle:
Working method:
Get reaction reagent 3mL, enzyme liquid/water 2mL mixes, and reacts 5min under 37 degree conditions, and reaction system is carried out mass spectrometric detection.Wherein SAM has specific 399 spectrum peak, and the size of 399 peak areas determines whether to respond in contrast blank and the sample, thereby determines methyl transferase activity.
To sum up, the present inventor has obtained a kind of homocysteine methyltransgerase, it derives from Candida albicans (Candida albicans SC5314), and molecular weight is 36KD, iso-electric point: 5.39, have the nucleotide sequence (such as Fig. 1) shown in the aminoacid sequence (such as Fig. 2) shown in the SEQ ID NO:1 and the SEQ ID NO:2.And homocysteine methyltransgerase of the present invention still has relative reactivity more than 60% at 40 ℃ of lower 15min, and its thermostability is the homocysteine methyltransgerase of inactivation significantly better than existing 40 ℃.Wherein, those skilled in the art are by reading present embodiment, can know for example SEQ ID NO:2 of nucleotide sequence of the present invention, and can obtain described nucleotide sequence by chemical synthesis process, therefore can be after obtaining having the nucleic acid of this nucleotide sequence, need not to implement present embodiment A) step, but after obtaining the nucleotide sequence shown in the SEQ ID NO:2 of synthetic, directly implement present embodiment step B)-E) repeat present embodiment; Perhaps obtaining step D) directly implement present embodiment step D behind e. coli bl21 (DE3) bacterial strain of described homocysteine methyltransgerase) and E) repeat present embodiment.
Preparation Example 2 heat-resisting sudden change homocysteine methyltransgerase screening amplification and mensuration
With PCR method gene order is carried out random mutation, condition:
10 μ l 10 * sudden change PCR damping fluid (70mmol/L MgCl2,500mM KCl, the 100mmol/L Tris-HCl of pH8.3,0.1% (w/v) gelatin), 50 μ M dATP, 50 μ M dGTP, 50 μ M dTTP, 50 μ M dCTP, 400nM primer SAMF, 400nM primer SAMR, 1.5U Pfu archaeal dna polymerase (Promega, USA), 20ng genomic dna (being the DNA with the nucleotide sequence shown in the SEQ ID NO:2 that embodiment 1 obtains) adds reaction system, transfers to reaction volume with sterilized water and reaches 100 μ L.The amplified reaction program is: above-mentioned reaction system 95 ℃ of lower reactions 5 minutes, is then carried out " 95 ℃ 30 seconds, 55 ℃ 30 seconds and 72 ℃ 2 minutes " of 30 circulations, at last 72 ℃ of lower maintenances 10 minutes.The pcr amplification gene product that obtains is separated with 1% agarose gel electrophoresis, and reclaim the dna fragmentation of the single band in the 936bp left and right sides with QIAGEN rapid extraction gel reagents box.
The PCR fragment that obtains through NcoI and XhoI double digestion 4h, through the pET30a carrier of NcoI and XhoI double digestion 4h, is connected the two by using the T4DNA ligase enzyme with equally, be built into recombinant expression vector, be converted in the e. coli bl21 (DE3).
Clone obtained above is inoculated into respectively in the LB substratum that contains kalamycin resistance, 37 ℃ are cultured to logarithmic phase, after inducing 4h with the IPTG of 1mM under 30 ℃ of conditions, with ultrasonic disruption cell (broken power 200w, broken time 4min) after, respectively at 40 ℃, 45 ℃, 50 ℃ lower heating in water bath 15min.
The ultrasonic cell-break liquid of above-mentioned heating is measured enzyme according to Preparation Example 1 described method lives.3 holes that have enzyme to live are arranged in 40 ℃ of enzyme liquid that heated, 1 hole that has enzyme to live is arranged, the hole that in 50 ℃ of enzyme liquid that heated, does not have enzyme to live in 45 ℃ of enzyme liquid that heated.The order-checking check is described behind 45 ℃ of heating ultrasonic cell-break liquid, the corresponding clone of institute in the hole that enzyme lives is arranged, the nucleotide sequence (shown in SEQ ID No.:4) of the heat-resisting sudden change homocysteine methyltransgerase that obtains encoding and express as described in the e. coli bl21 (DE3) of heat-resisting sudden change homocysteine methyltransgerase.
The method of putting down in writing according to Preparation Example 1, obtain heat-resisting homocysteine methyltransgerase, it derives from Candida albicans (Candida albicans SC5314), and molecular weight is 36KD, iso-electric point: 5.39, have the nucleotide sequence shown in the aminoacid sequence shown in the SEQ ID NO:3 and the SEQ ID NO:4.And present embodiment gained homocysteine methyltransgerase still all has the relative reactivity more than 50% after leaving standstill 15 minutes under 45 ℃, its thermostability is the homocysteine methyltransgerase of inactivation significantly better than existing 40 ℃ not only, and is better than the type methyltransferase of embodiment 1.
Test implementation example 1
Test as follows the thermostability of the homocysteine methyltransgerase of homocysteine methyltransgerase that Preparation Example 1 do not suddenly change and Preparation Example 2 sudden changes:
It is in 7.5 the 20mmol/L potassium phosphate buffer that the homocysteine methyltransgerase dry powder of 1KU is dissolved in the pH value, respectively 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, lower maintenance 15 minutes.Measuring enzyme according to the method for Preparation Example 1 lives.The ratio percentage ratio of living with the maximum institute enzyme of surveying work by the survey enzyme is that relative reactivity is ordinate zou, and temperature is the X-coordinate curve plotting.Preparation Example 1 and 2 test result are seen Fig. 3.
As can be seen from the figure, the thermostability of the homocysteine methyltransgerase of the homocysteine methyltransgerase that Preparation Example 1 is not suddenlyd change and Preparation Example 2 sudden changes is all significantly greater than the thermostability (not shown on the figure, storage temperature is complete deactivation above 40 ℃) of the homocysteine methyltransgerase of prior art.As shown in Figure 3, two kinds of homocysteine methyltransgerase of Preparation Example 1-2 still all have the relative reactivity more than 60% after leaving standstill 15 minutes under 40 ℃, wherein, the homocysteine methyltransgerase of Preparation Example 2 still all has the relative reactivity more than 50% after leaving standstill 15 minutes under 45 ℃.
Preparation Example 3-6
Formulated reagent one and reagent two according to table 1:
Table 1
Preparation Example 3 and 4 reagent one and reagent two are mixed with respectively liquid form and get final product.Preparation Example 5 and 6 reagent one and reagent two respectively under the following conditions lyophilizes: in general refrigerator-10 ℃ lower freezing 2 hours, and then-40 ℃ of cryogenic refrigerator pre-freezes 8 hours, in the ALPHA 1-4LSC of German Ke Ruisite (CHRIST) type freeze drier, with the condition freeze-drying of vacuum tightness 0.04mbar, safe pressure 0.100mbar and condenser temperature-60 ℃ 25 hours.Then waiting temperature of charge and Freeze Drying Equipment baffle temperature poor is zero, and observes 15 seconds internal pressures and indicate when constant, finishes freeze-drying.The reagent dry powder that freeze-drying obtains can redissolve into the volume of primary liquid form before use with distilled water.The reagent dry powder that freeze-drying obtains, longer than the time that the reagent of liquid form is preserved, packing instructions are lower, and volume is less, and it is easier to transport.
Preparation Example 3-6 has the calibration solution that homotype semicystinol concentration investigating is 34 μ mol/L.
Test implementation example 2
This test implementation example has been measured two samples to be measured: one is the homocysteine aqueous standard product of 9 μ mol/L of preparation; Another is 30 years old women's being in a good state of health serum sample to be measured, extracts its 10ml blood, leaves standstill to the hemocyte precipitated and separated, gets upper serum 5ml and is used for the test kit of Preparation Example 3-6 is tested.
Respectively the R1 among the Preparation Example 2-5 is got 240 μ l, add sample 13 μ l, sample and R1 add R2 reagent 65 μ l 37 ℃ of insulations 300 seconds.R1, R2 and sample mixed solution are incubated 60 seconds under 37 ℃ of conditions, record absorbancy numerical value A1 under the 340nm condition, and 180 seconds record absorbancy numerical value A2 are according to the variation of absorbancy numerical value.Test result sees Table 2.
Table 2
As can be seen from Table 2, no matter be the serum specimen that detects the preparation sample of concentration known or detect complicated component, the standard deviation of test kit detected result of the present invention is all very little, and is all very approaching with actual value, therefore they have all proved test kit stable performance of the present invention, measure accurately.
Claims (9)
1. a homocysteine methyltransgerase is characterized in that, the aminoacid sequence of described homocysteine methyltransgerase is the aminoacid sequence shown in the SEQ ID NO:3.
2. the nucleotide sequence of the homocysteine methyltransgerase of encoding is characterized in that, described nucleotides sequence is classified the nucleotide sequence of coding homocysteine methyltransgerase claimed in claim 1 as.
3. nucleotide sequence according to claim 2, wherein, described nucleotides sequence is classified as
Nucleotide sequence shown in the SEQ ID NO:4.
4. a recombinant vectors is characterized in that, described recombinant vectors is comprised of the goal gene of empty carrier and this empty carrier of insertion, and described goal gene is the nucleotide sequence of coding homocysteine methyltransgerase claimed in claim 2.
5. recombinant vectors according to claim 4 is characterized in that, described empty carrier is selected from the vehicle group that is comprised of pET serial carrier, pUC19, pGEM and pBluescript.
6. a recombinant host cell is characterized in that, described recombinant host cell contains recombinant vectors claimed in claim 4.
7. recombinant host cell according to claim 6 is characterized in that, described host cell is e. coli bl21 (DE3), BL21, TOP10 or DH5a.
8. an Accessory Right requires the method for purifying homocysteine methyltransgerase in the 6 described recombinant host cells, it is characterized in that, described method adopts the broken liquid with reconstitution cell to heat 15-40 minute at 40-45 ℃, then at the centrifugal 10-20min of 8000-15000rpm, to remove heat labile foreign protein.
9. a test kit that detects homocysteine is characterized in that, described test kit comprises
Homocysteine methyltransgerase claimed in claim 1.
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