CN1338466A - Polypeptide with alpha-galactosidase activity and its coding nucleic acid - Google Patents
Polypeptide with alpha-galactosidase activity and its coding nucleic acid Download PDFInfo
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- CN1338466A CN1338466A CN00122736A CN00122736A CN1338466A CN 1338466 A CN1338466 A CN 1338466A CN 00122736 A CN00122736 A CN 00122736A CN 00122736 A CN00122736 A CN 00122736A CN 1338466 A CN1338466 A CN 1338466A
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Abstract
A polypeptide with higher alpha-galactosidase activity, the polynucleotide for coding it, the nucleic acid configurator, carrier and host cell, which contain said polynucleotide, the process for preparing said polypeptide, the application of said polypeptide in B to O blood type transform, and the method for B to O blood type transform are disclosed.
Description
The present invention relates to a kind of isolated nucleic acid sequences with new polypeptide and this polypeptide of encoding of alpha-galactosidase activity.The present invention also relates to comprise nucleic acid construct, carrier and the host cell of this nucleotide sequence and the method for preparing this polypeptide.The invention further relates to the purposes of this polypeptide in B → O blood group conversion.The invention still further relates to a kind of B → O blood group conversion method.
Abo blood group is the of paramount importance blood group system of clinical meaning, if thereby blood group is not inconsistent and can causes that acute haemolysis causes death during blood transfusion.In general, O type blood is universal blood, can be defeated by the patient of any blood group.If the blood of other blood group can be changed into the O type, can make full use of blood resource undoubtedly, reduce the blood transfusion accident, simplify the blood transfusion program, shorten the waiting time, more great to the blood transfusion first aid meaning under the emergency situation.
Human A, B, O blood group are determined as antigenic sugar chain that by erythrocyte surface these structures correspondingly are called A, B, H antigen.B antigen is compared with H antigen, and internal core is identical, and difference only is the α-galactose residue that the former least significant end has more.Under the effect of alpha-galactosidase activity, α-galactose residue the hydrolysis of Type B red corpuscle least significant end is removed, just can be transformed into H antigen to B antigen, thereby change Type B blood into the general blood of O type (Yatziv et al, 1971, Biochem.Biophys.Res.Commun, 45,514-518).
Alpha-galactosidase (α-galactosidase, EC 3.2.1.22) be extensively to be present in the circumscribed glycoside hydrolase of the intravital class of people, animal, plant, fungi and prokaryotic organism (Corcheteet al, 1987, Phytochemistry, 26,927-932), but the α-galactose residue of various polysaccharide substrate ends is removed in the hydrolysis of high degree of specificity ground, has vital role in the life Metabolic activity.
As far back as the 60 to 70's, people just find that alpha-galactosidase has the activity of hydrolysis blood group antigen material, and recognize that this characteristic of utilizing this enzyme can realize the external transformation to human blood group.
So far, around different plant species source, particularly the alpha-galactosidase of plant origin has carried out big quantity research, but because the biochemical property difference is not that all alpha-galactosidases are applicable to that all B → O blood group changes.Wherein, the coffee berry alpha-galactosidase be considered to optimal selection (people such as Harpaz, 1975, Arch.Biochem.Biophys, 170,676-683).Bibliographical information is arranged, exist in the different sorts coffee berry alpha-galactosidase of various ways isozyme (people such as Courtois, 1966, Methods in Enzymol, 8,565-571).
Disclose in United States Patent (USP) 4330619 (May 18 nineteen eighty-two) and 4427777 (on January 24th, 1984) and utilized the alpha-galactosidase that from green Santos coffee berry, extracts that HRBC Type B antigenic shift is the antigenic method of H type, thereby prove that this fermentoid has the function that B → O blood group changes, but the method for practicability is failed to provide reliably, is enough to above-mentioned document.In addition, the alpha-galactosidase that extracts from plants such as coffee berry contains compositions such as tannin, pigment, and alpha-galactosidase activity is had restraining effect, its practical application (Goldsteinet al, 1965, Phytochemistry have been limited more, 4,185-192).
USA New York Blood Center Goldstein etc. utilizes above-mentioned Santos coffee berry alpha-galactosidase to carry out B → O blood group transformation research, and carried out the human clinical in 1996 through drugs approved by FDA and test, but gap is still arranged apart from practical application, major cause is, the specific activity of Santos coffee berry alpha-galactosidase is lower, be about 30U/mg, enzymolysis changes the enzyme that 1ml Type B red corpuscle needs 180-200U, unit red corpuscle then need about 1-2g, and consumption is big, cost is high, is difficult to practicability (Lenny, 1991, Blood, 77,1383-1388).In addition, the alpha-galactosidase quantity of natural origin is extremely limited, and the enzyme amount of extracting from 50 pounds of Santos coffee berrys only can satisfy a blood group and change the needs of testing.Therefore, in clinical, press for a kind of specific activity alpha-galactosidase activity polypeptide higher, that easily prepare.
One object of the present invention is to provide a kind of polypeptide with higher alpha-galactosidase activity.Another object of the present invention is to provide the polynucleotide of coding said polypeptide.Another object of the present invention is to provide the nucleic acid construct that contains described polynucleotide, contains the expression vector and the host cell of this nucleic acid construct.The present invention also aims to provide preparation method, the purposes of this polypeptide, and a kind of B → O blood group conversion method.
The present invention relates to a kind of isolated polypeptide with alpha-galactosidase activity, it is characterized in that described polypeptide has the aminoacid sequence identical with aminoacid sequence shown in the SEQ ID NO:2 at least 80%, the alpha-galactosidase activity of this polypeptide is higher than having shown in the SEQ ID NO:3 more than the sequence alpha-galactosidase activity of peptide at least.
The invention still further relates to the isolating polynucleotide of coding said polypeptide and comprise the method for nucleic acid construct, carrier and host cell and the preparation and the described polypeptide of application of these polynucleotide.
Fig. 1 has shown middle fruit coffee Bourbon Santos Cultivar (Coffea canephora cv.Bourbon Santos, hereinafter referred Santos) comparison of the alpha-galactosidase gene cDNA sequence of coffee berry and fruitlet coffee Catimor Cultivar (Coffea arabica cv.Catimor, hereinafter referred Catimor) coffee berry.Wherein line part is depicted as the coding region, and amino-acid sequence is calculated with first amino acid of the whole protein that contains signal peptide.
Fig. 2 A, 2B have shown the structure of the carrier pPICZ α A that is used for making up Catimor coffee berry alpha-galactosidase expression vector and constructed expression vector pPICZ-Gal.
Fig. 3 illustrates in 7 days process of cultured continuously, the measurement result of alpha galactosides specific enzyme activity in the recombinant bacterial strain culture supernatant.
The purity and the protein molecular weight of purified product alpha-galactosidase measured in Fig. 4 explanation with SDS-PAGE.
Fig. 5 illustrate the Type B red corpuscle after alpha-galactosidase enzyme is separated 2 hours with the serology aggregation situation of various antibody.A, B represent the anti-A of standard, the anti-B antibody that uses clinically respectively among the figure; And 1024,512 ..., the anti-B monoclonal antibodies of representing the high titre of different extension rates respectively such as 4,2.
In first embodiment, the present invention relates to have the polypeptide of the separation of alpha-galactosidase activity, it is characterized in that:
(1) described polypeptide has the amino acid sequence identical with amino acid sequence shown in the SEQ ID NO:2 at least 80%, and
(2) alpha-galactosidase activity of this polypeptide is at least than having shown in the SEQ ID NO:3 The alpha-galactosidase activity of peptide is higher more than the sequence.
Preferably, the same degree of sequence shown in described amino acid sequence of polypeptide and the SEQ ID NO:2 is about at least 90%, more preferably is about 95% at least, most preferably about at least 97% (being referred to herein as " homeopeptide ").In a preferred embodiment, the aminoacid sequence that this homeopeptide has has 5 amino acid different with SEQ ID NO:2 aminoacid sequence, preferred 4 amino acid differences, more preferably 3 differences, also preferably 2 differences, most preferably 1 amino acid difference.For the purposes of the present invention, same degree between two or more aminoacid sequences is by BLAST2.0 queries of protein databases program (Altschul etc., 1997, nucleic acids research 25:3389-3402) and adopt following parameter to determine: blastall-pblastp-a4-e10-E0-v500-b250-I[inquires about document]-d prot_all, wherein-p refers to program name,-a refers to the server count that will use,-e refers to expected value,-E refers to extend the cost of breach,-v refers to single line description (one-line description) number,-b refers to the ratio logarithm that will show ,-I refers to inquire about document, and-d refers to the database that is used to inquire about.
Preferably, polypeptide of the present invention comprises the aminoacid sequence of SEQ ID NO:2; Or it has alpha-galactosidase activity and activity level at least than having shown in the SEQ ID NO:3 the higher fragment of alpha-galactosidase activity of peptide more than the sequence.In a further preferred embodiment, polypeptide of the present invention comprises the aminoacid sequence of SEQ ID NO:2.In another preferred embodiment, polypeptide of the present invention is by the aminoacid sequence of SEQ ID NO:2, and perhaps it has alpha-galactosidase activity and activity level is formed than having the fragment that the alpha-galactosidase activity of peptide is higher more than the sequence shown in the SEQ ID NO:3 at least.
The fragment of SEQ ID NO:2 be the N-terminal of this aminoacid sequence and/or carboxyl-terminal deletion one or more amino acid whose polypeptide.Preferably, fragment contains at least 300 amino-acid residues, and more preferably at least 325 amino-acid residues most preferably have at least 350 amino-acid residues.
The aminoacid sequence difference of the aminoacid sequence of homeopeptide and SEQ ID NO:2 or its mature polypeptide may be to insert, add and/or lack 1 or a plurality of amino-acid residue and/or have 1 or a plurality of amino-acid residue replaced by the different aminoacids residue.Preferably, amino acid change is that character changes less variation, promptly be can the remarkably influenced Protein Folding and/or active conservative amino acid replace; The small segment disappearance, normally 1 to about 30 amino acid whose disappearances; Little amino or C-terminal extend, as the methionine residues of aminoterminal interpolation; The little connection peptides that reaches about 20-25 residue is arranged; Maybe can be by changing little extension such as poly Histidine fragment, epitope or the land that net charge or other function help purifying.
The example that conservative property replaces is the replacement of carrying out in basic aminoacids (arginine, Methionin and Histidine), acidic amino acid (L-glutamic acid and aspartic acid), polare Aminosaeren (glutamine and l-asparagine), hydrophobic amino acid (leucine, Isoleucine and Xie Ansuan), die aromatischen Aminosaeuren (phenylalanine, tryptophane and tyrosine) and p1 amino acid (glycine, L-Ala, Serine, Threonine and methionine(Met)).The aminoacid replacement that can not change specific activity usually is known in the art, and by for example H.Neurath and R.L.Hill, 1979, at " protein " book, Academic Press described among the New York.Modal replacement is Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn,, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly and the replacement that is reversed.
The alpha-galactosidase activity of polypeptide of the present invention is high more about 25% than having shown in the SEQ ID NO:3 more than the aminoacid sequence alpha-galactosidase activity level of peptide at least, and is preferably high at least about 50%, more preferably high at least by about 75%, most preferably high at least about 99%.
The present invention relates to a kind of isolated polypeptide with alpha-galactosidase activity in addition, it is characterized in that:
(1) described polypeptide has the aminoacid sequence identical with aminoacid sequence shown in the SEQ ID NO:2 at least 80%, and
(2) with this polypeptide and p-nitro-benzene base-α-D-galactopyranoside 1.25mmol/L, pH6.5 mixes, and 26 ℃ of incubations are after 1 hour, and the alpha-galactosidase activity that records is higher than 30U/mg at least.
In a preferred embodiment, the alpha-galactosidase activity of this polypeptide is at least about 40U/mg.In a preferred embodiment, the alpha-galactosidase activity of this polypeptide is at least about 50U/mg.In another preferred embodiment, the alpha-galactosidase activity of this polypeptide is at least about 62U/mg.
In a preferred embodiment of the invention, polypeptide of the present invention can derive from fruitlet coffee, especially fruitlet coffee Catimor Cultivar.
Term " can derive from " in the context of the invention, refer to the polypeptide with alpha-galactosidase activity of the present invention can be by fruitlet coffee, preferred fruitlet coffee Catimor Cultivar produces, or can be produced by the cell of the gene of peptide more than the coding tool alpha-galactosidase activity that has inserted this source.
In a more preferred, polypeptide of the present invention has the aminoacid sequence of SEQ ID NO:2.
Should be understood that above-mentioned species, the present invention includes other taxonomy Equivalent, and no matter its known species title how.Those skilled in that art can recognize the consistence of corresponding equivalence name easily.
The polypeptide of the present invention's nucleic acid sequence encoding also is included in polypeptide or its segmental N-end or C-end and has merged the fusion polypeptide of another polypeptide or the fusion polypeptide of cleavable.The nucleotide sequence (or its part) of another polypeptide of coding just can be produced fusion polypeptide with nucleotide sequence of the present invention (or its part) fusion.The technology that produces fusion polypeptide is known in the art, comprise the encoding sequence that connects coded polypeptide, thereby make them in same frame, and the expression of fusion polypeptide is controlled by identical promotor and terminator.Polynucleotide
The invention still further relates to the separation polynucleotide of the nucleotide sequence that contains the polypeptide of the present invention of encoding.In a preferred embodiment, these polynucleotide comprise the nucleotide sequence of SEQ ID NO:1.In another preferred embodiment, described polynucleotide comprise the nucleotide sequence that is had among the contained plasmid pPICZ-Gal in the pichia pastoris CGMCC No.0479.The present invention comprises that also coding has the nucleotide sequence of polypeptide of SEQ ID NO:2 aminoacid sequence, and is different because of the degeneracy of genetic code between it and the SEQ ID NO:1.The invention still further relates to the polynucleotide of the subsequence that comprises SEQ ID NO:1, have alpha-galactosidase activity and the activity level of described subsequence coding SEQ ID NO:2 are to have shown in the SEQ ID NO:2 75% of the alpha-galactosidase activity level of peptide fragment more than the sequence at least.Nucleotide sequence of the present invention comprises genome sequence, and corresponding cDNA and RNA sequence.Term used herein " nucleotide sequence " is understood to include synthetic DNA in all interior these class mutation.
The subsequence of SEQ ID NO:1 is the nucleotide sequence that SEQ ID NO:1 is contained, but has fallen 1 or a plurality of Nucleotide from 5 ' terminal and/or 3 ' terminal deletion.Preferably, subsequence contains at least 900 Nucleotide, more preferably at least 975 Nucleotide, most preferably at least 1050 Nucleotide.
The invention still further relates to the separation polynucleotide that contain the mutant nucleic acid sequence that in the polypeptid coding area of SEQ ID NO:1, has a sudden change at least, wherein the polypeptide shown in this mutant nucleic acid sequence encoding SEQID NO:2.
The invention still further relates to the coding active polypeptide, the nucleotide sequence of certain homology arranged with the mature polypeptide encoded sequence of SEQ ID NO:1, the homology degree is at least about 70%, preferred about 80%, more preferably from about 90%, also will be preferably about 95%, most preferably about 97% homology.With regard to the object of the invention, the homology degree between two nucleotide sequences is by Wilbur-Lipman method (Wilbur ﹠amp; Lipman, 1983, the journal 80:726-730 of NAS) uses LASERGENE
TMMEGALIGN
TMSoftware (DNASTAR, Inc., Madison, WI) and homogeny table and following multiple reduced parameter: breach point penalty and notch length point penalty are 10 and determine.Reduced parameter is Ktuple=3 in pairs, breach point penalty=3, window (windows)=20.
During the similar substantially polypeptide of synthetic and polypeptide of the present invention, have necessity the nucleotide sequence of code book invention polypeptide is modified.Term and described polypeptide " similar substantially " are meant the polypeptide of non-natural form.These polypeptide may be different on some processing mode with the polypeptide that is separated to from natural origin.For example, may wish to synthesize variant polypeptides with for example site-directed mutagenesis, these variants have different thermostabilitys or optimal pH etc.Can for example construct similar sequence on its subsequence basis at the nucleotide sequence of peptide encoding part more than the SEQ ID NO:1; And/or replace and make up by introducing Nucleotide, described replacement can not make the aminoacid sequence of generation different with former nucleotide sequence encoded polypeptides, but they meet enzyme and prepare the use habit of used host living beings to codon; Or replace by the Nucleotide that introducing can produce the different aminoacids sequence and to make up.The summary that replaces about Nucleotide, referring to for example Ford etc., 1991, protein expression and purifying 2:95-107.
It will be apparent for a person skilled in the art that and beyond the molecular function key area, to do these replacements, and still can obtain active polypeptide.Those for activity of the coded polypeptide of separated nucleic acid sequence of the present invention must, so the amino-acid residue that does not preferably replace can be according to the known operation of this area, for example site-directed mutagenesis or alanine scanning mutagenesis are (referring to for example, Cunningham and Wells, 1989, science 244:1081-1085) identify.In a kind of technology in back, in molecule, import sudden change on each positively charged residue, thereby the amino-peptidase activity that detects the gained mutating molecule then identifies the amino-acid residue relatively more crucial to molecular activity.By to analyzing with measuring three-dimensional structure as nucleus magnetic resonance, crystallography or light affinity tag, can determine substrate-enzyme interacting the site (referring to for example, de Vos etc., 1992, science 255:306-312; Smith etc., 1992, molecular biology magazine 224:899-904; Wlodaver etc., 1992, FEBS wall bulletin (FEBS Letters) 309:59-64).Nucleic acid construct
The invention still further relates to the nucleic acid construct of 1 or a plurality of regulating and controlling sequences that comprise nucleotide sequence of the present invention and can be operatively connected with it, described regulating and controlling sequence can instruct encoding sequence to express in proper host cell under its consistency condition.Expression be understood to include polypeptide produce in related any step, include, but are not limited to transcribe, post transcriptional modificaiton, translation, posttranslational modification and secretion.
" nucleic acid construct " is defined as strand or double chain acid molecule in the text, and they separate from natural gene, and be perhaps modified and contain in the non-natural mode and make up and nucleic acid fragment arranged side by side.When nucleic acid construct comprises when expressing essential all regulating and controlling sequences of encoding sequence of the present invention term nucleic acid construct and expression cassette synonym.Term " encoding sequence " is defined as the part of directly determining the aminoacid sequence of its protein product in the nucleotide sequence in the text.The border of encoding sequence is normally determined by the ribosome bind site (for prokaryotic cell prokaryocyte) of next-door neighbour mRNA 5 ' end opening code-reading frame upstream and the transcription termination sequence in next-door neighbour mRNA 3 ' end opening code-reading frame downstream.Encoding sequence can include, but are not limited to DNA, cDNA and recombinant nucleic acid sequence.
Can operate the isolated nucleic acid sequences of coding polypeptide of the present invention in many ways, make it express described polypeptide.May expect or must before inserting carrier, process that this depends on expression vector to nucleotide sequence.The technology of using recombinant DNA method modification of nucleic acids sequence is known in the art.
Herein term " control sequence " be defined as comprise express polypeptide of the present invention institute must or favourable all components.Each regulating and controlling sequence can be natural that contain or external for the nucleic acid encoding sequence.These regulating and controlling sequences include, but not limited to leader sequence, polyadenylic acid sequence, propeptide sequence, promotor, signal sequence and transcription terminator.Bottom line, regulating and controlling sequence will comprise promotor and the termination signal of transcribing and translating.So that the coding region of regulating and controlling sequence with the nucleic acid encoding sequence is connected, can provide the regulating and controlling sequence of belt lacing in order to import specific restriction site.Term " can be operatively connected " and be defined as a kind of like this conformation in the text, and wherein regulating and controlling sequence is positioned at the appropriate location of the encoding sequence of relative dna sequence dna, so that regulating and controlling sequence instructs polypeptide expression.
Regulating and controlling sequence can be suitable promoter sequence, can be by the nucleotide sequence of the host cell of express nucleic acid sequence identification.Promoter sequence contains the transcription regulating nucleotide sequence that mediates expression of polypeptides.Promotor can be any nucleotide sequence that transcriptional activity is arranged in selected host cell, comprises sudden change, brachymemma and promotor heterozygosis, can get the gene of polypeptide in the outer or born of the same parents of own coding and host cell homology or allogenic born of the same parents.
Regulating and controlling sequence can also be suitable transcription termination sequence, thereby can be discerned one section sequence that termination is transcribed by host cell.Terminator sequence can be operatively connected 3 ' end in the nucleic acid encoding sequence.Any terminator that can bring into play function in selected host cell may be used to the present invention.
Regulating and controlling sequence can also be suitable leader sequence, promptly to the crucial mRNA non-translational region of the translation of host cell.Leader sequence can be operatively connected 5 ' end in the nucleic acid encoding sequence.Any leader sequence that can bring into play function in selected host cell all can be used for the present invention.
Regulating and controlling sequence can also be a signal peptide coding region, and this district's coding is connected in the N-terminal aminoacid sequence of polypeptide for one section, can guide coded polypeptide to enter the emiocytosis approach.5 ' the end in nucleic acid sequence encoding district may be natural contain the signal peptide coding region that the translation frame as one man is connected naturally with the coding region fragment of secrete polypeptide.Perhaps, can to contain encoding sequence be external signal peptide coding region to 5 ' of coding region end.When encoding sequence does not under normal circumstances contain signal peptide coding region, may need to add the extraneous signal peptide-coding region.Perhaps, can replace the natural signals peptide-coding region simply to strengthen the polypeptide secretion with external signal peptide coding region.But the signal peptide coding region that the polypeptide after any energy guiding is expressed enters the Secretory Pathway of used host cell may be used to the present invention.
Regulating and controlling sequence can also be peptide original encoding district, and this district's coding is positioned at the aminoterminal one section aminoacid sequence of polypeptide.The gained polypeptide is called as proenzyme or propolypeptide.Propolypeptide does not have activity usually, can be by catalysis or self-catalysis and be converted into sophisticated active polypeptide from propolypeptide cutting peptide is former.
When the existing signal peptide of the N-terminal of polypeptide has the former district of peptide again, the N-terminal of peptide former district next-door neighbour polypeptide, the signal peptide district then is close to the N-terminal in the former district of peptide.
The regulating and controlling sequence that interpolation can be regulated expression of polypeptides according to the growing state of host cell may also be needs.The example of regulator control system is that those can be reacted to chemistry or physical stimulation thing (being included under the situation of regulating compound), thereby opens or close the system of genetic expression.Other examples of regulating and controlling sequence are those regulating and controlling sequences that can make gene amplification.In these examples, nucleic acid encoding sequence and regulating and controlling sequence can should be operatively connected together.Expression vector
The invention still further relates to and comprise nucleotide sequence of the present invention, promotor and transcribe and the recombinant expression vector of translation termination signal.Above-mentioned various nucleic acid and regulating and controlling sequence can be linked together prepares recombinant expression vector, and this carrier can comprise 1 or a plurality of restriction site easily, so that insert in these sites or replace the nucleic acid encoding sequence.Perhaps, can express nucleotide sequence of the present invention by nucleotide sequence or the nucleic acid construct that comprises this sequence are inserted suitable expression vector.Preparation is during expression vector, can make encoding sequence be arranged in carrier so that can be operatively connected with suitable expression regulation sequence.
Recombinant expression vector can be any carrier (for example plasmid or virus) of being convenient to carry out recombinant DNA operation and express nucleic acid sequence.The consistency of the host cell that carrier and it will import is depended in the selection of carrier usually.Carrier can be linearity or closed loop plasmid.
Carrier can be self-replicating type carrier (promptly be present in extrachromosomal complete structure, can be independent of karyomit(e) and duplicate), for example plasmid, extra-chromosomal element, minute chromosome or artificial chromosome.Carrier can comprise any mechanism that guarantees self-replacation.Perhaps, carrier be one when importing host cell, with the carrier that is incorporated in the genome and duplicates with the karyomit(e) that is incorporated into.In addition, can use single carrier or plasmid, or totally comprise and will import the two or more carriers or the plasmid of all DNA of host cell gene group, or transposon.
Preferred carrier of the present invention contains 1 or a plurality of selective marker of being convenient to select transformant.Selective marker is such gene, and its product is given to the resistance of biocide or virus, to the resistance of heavy metal, or gives auxotroph prototroph etc.The dal gene of the example of bacterium selective marker such as subtilis or Bacillus licheniformis, the perhaps resistance marker of microbiotic such as penbritin, kantlex, paraxin or tsiklomitsin.
Preferred carrier of the present invention comprises can make the carrier stable integration in the host cell gene group, or guarantees carrier to be independent of cellular genome in cell and carry out the element of self-replicating.
With regard to the situation that is incorporated into the host cell gene group, carrier can depend on other elements of nucleic acid encoding sequence or carrier, so that carrier stably is incorporated in the genome by homology or non-homogeneous reorganization.Perhaps, carrier can contain and is useful on guiding and is incorporated into additional nucleotide sequence in the host cell gene group by homologous recombination.Additional nucleotide sequence can make vector integration accurate site on the karyomit(e) in the host cell gene group.In order to be increased in the possibility that accurate site is integrated, preferred integrated element should contain the nucleic acid of sufficient amount, as 100 to 1500 base pairs, preferred 400 to 1500 base pairs, 800 to 1500 base pairs most preferably, and with respective target sequence height homology, to increase the possibility of homologous recombination.Integrated element can be and hit any sequence of sequence homology of host cell gene group.In addition, integrated element can be non-coding or nucleic acid sequence encoding.On the other hand, carrier can be by non-homogeneous recombination and integration in the genome of host cell.
With regard to the situation of carrying out self-replicating, carrier can also comprise replication orgin, and carrier can independently be duplicated in target host cell.Replication orgin can have makes its sudden change that becomes responsive to temperature type in host cell (referring to for example, Ehrlich, 1978, the journal 75:1433 of NAS).
Can insert the nucleotide sequence of the present invention of copy more than 1 to improve the output of this gene product to host cell.The copy number increase of this nucleotide sequence can be by inserting 1 additional copies of this sequence in the host cell gene group at least, perhaps insert a selective marker that can increase with this nucleotide sequence, by culturing cell in the presence of the suitable selective reagents is being arranged, thereby pick out the cell that the selectable marker gene that containing the amplification copy contains the additional copies nucleotide sequence.
Be used to connect operation that above-mentioned each element makes up recombinant expression vector of the present invention and be well known to those skilled in the art (referring to for example, Sambrook etc., molecular cloning laboratory manual, second edition, press of cold spring harbor laboratory, cold spring port, New York, 1989).Host cell
The invention still further relates to the recombinant host cell that comprises the nucleotide sequence of the present invention that can be used to the recombinant production polypeptide.The carrier that comprises the present invention's nucleotide sequence can be imported host cell, thereby this carrier is maintained with the outer carrier format of the karyomit(e) of above-mentioned chromosomal integration body or self-replacation.Term " host cell " is contained the sudden change that takes place between any because replicative phase and the offspring different with parental cell.Peptide coding gene and source thereof are depended in the selection of host cell to a great extent.
Host cell can be prokaryotic cell prokaryocyte or eukaryotic cell, preferred eukaryotic cell, especially pichia pastoris.Can carrier be imported host cell by technology well known to those skilled in the art.
The preparation method
The invention still further relates to the method for preparing polypeptide of the present invention, this method comprises from fruitlet coffee, especially this polypeptide of purifying in the fruitlet coffee Catimor Cultivar.
The invention still further relates to recombinates prepares the method for polypeptide of the present invention, and this method comprises: (a) be suitable for producing under the condition of this polypeptide, cultivating the host cell that contains nucleic acid construct, this nucleic acid construct comprises the nucleotide sequence of this polypeptide of encoding; (b) reclaim this polypeptide.
In preparation method of the present invention, with means known in the art culturing cell in the nutritional medium that is fit to the polypeptide generation.For example, can be in suitable medium, allowing under expression of polypeptides and/or the isolating condition, come culturing cell by shake-flask culture, laboratory or industrial fermentation jar middle and small scale or large scale fermentation (comprise continuously, in batches, batch charging or solid state fermentation).In the suitable medium that comprises carbon and nitrogenous source and inorganic salt, adopt step known in the art to cultivate.Suitable medium can be provided or can be prepared with reference to disclosed the composition (for example, described in the catalogue of American type culture collection) by supplier.If polypeptide is secreted in the substratum, then can directly from substratum, reclaim polypeptide.If polypeptide is not secreted, can from cell lysate, reclaim.
Can detect polypeptide with the special method at described polypeptide known in the art.These detection methods comprise the use of specific antibody, the formation of enzyme product or the disappearance of enzyme substrates.For example, can determine the activity of polypeptide with enzyme process.The step that is used to measure alpha-galactosidase activity is known in the art.A kind of method of measuring alpha-galactosidase activity has above been described.
Can reclaim the polypeptide that is produced with means known in the art.For example, can from substratum, reclaim polypeptide by routine operation (including, but are not limited to centrifugal, filtration, extracting, spraying drying, evaporation or precipitation).
Can come purifying polypeptide of the present invention by various operations known in the art, these operations comprise, but (for example be not limited to chromatography, ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing and size exclusion chromatography), electrophoresis (for example, preparation property isoelectric focusing), differential solubleness (for example ammonium sulfate precipitation), SDS-PAGE or extracting (referring to for example, protein purification, J.C.Janson and Lars Ryden compile, VCH Publishers, NewYork, 1989).
In yet another embodiment of the present invention, the invention provides a kind of B → O blood group conversion method, this method may further comprise the steps:
(1) separate the Type B hemocyte,
(2) with suitable isotonic buffer solution washing Type B hemocyte,
(3) add any one described polypeptide among the claim 1-16,
(4) at 20-26 ℃ of incubation one to a few hours,
(5) use the phosphate buffered saline(PBS) washed cell.
In a preferred embodiment, described isotonic buffer solution is the PCS (60mMNaH of acid pH
2PO
4, 25mM Trisodium Citrate, 75mM sodium-chlor), described neutral isotonic buffer solution is PBS.
Further describe the present invention by following examples, but it should be interpreted as limitation of the scope of the invention.
Embodiment
(CA 92008 for 1600Faraday Avenue, Carlsbad, and the operational manual that USA) provides carries out according to Invitrogen company basically in relevant zymic operation among the following embodiment.
The clone of Catimor coffee berry alpha-galactosidase cDNA
Pluck green fresh Hainan and produce Catimor coffee berry ,-70 ℃ of preservations.The coffee berry that takes out-70 ℃ of preservations places the copper mortar of precooling, adds liquid nitrogen and is ground into powder rapidly.After tannin and pigment are removed in the acetone soln extracting, use RNAgents
TMTotal RNA isolationsystem (Promega company, 2800 Woods Hollow Road, Madison, WI53711-5399, USA) test kit extracts cell total rna: get about the coffee berry sample 50mg of extracting, add sex change liquid 600 μ l, mix in the ice bath, add 60 μ l2MNaAc (pH4.0) solution immediately, fully add the phenol of 600 μ l behind the mixing: chloroform: primary isoamyl alcohol solution (25: 24: 1), placed 15 minutes on ice; 4 ℃ of 10,000 * g are centrifugal 20 minutes; Water adds the equal-volume Virahol, and-20 ℃ precipitate 30 minutes; 4 ℃ of 10,000 * g are centrifugal 20 minutes; Ice-cold 75% ethanol thorough washing precipitation is placed to the water dissolution precipitation that adds an amount of nuclease free after the partial desiccation ,-20 ℃ of preservations standby (prolonged preservation then places RNA in the ethanol and precipitates ,-70 ℃ of preservations).Utilize uv-absorbing and denaturing gel electrophoresis to detect rna content, purity and integrity.
Consider the alpha-galactosidase of different varieties coffee berry, its gene order big difference that do not have, we are reference literature report Santos coffee berry alpha-galactosidase cDNA sequence (ZhuA.1994, gene, 140 (2), 227-231), outside the coding region, design primer, upstream primer is 5 '-CCGCTCGAGTTGGTGCTTCCGCT-3 ', downstream primer is 5 '-CGGAATTCTGCATTCTCCTG-3 ', adopt Access RT-PCR System (Promega, 2800 Woods Hollow Road, Madison, WI 53711-5399 USA) carries out reverse transcription PCR: mix AMV/Tfl 5x damping fluid 10 μ l in 50 μ l reaction systems, total about 1 μ g of RNA template, on, each 50pmol of downstream primer, each 1 μ l of reversed transcriptive enzyme and archaeal dna polymerase, dNTP mixture (each 10mM) 1 μ l, 25mM MgSO
42 μ l and suitable quantity of water, 48 ℃ of reverse transcription reactions 45 minutes, 94 ℃ of deactivation reversed transcriptive enzymes 2 minutes entered 94 ℃ 30 seconds, 62 ℃ 1 minute, 68 ℃ of PCR circulations 2 minutes, and 40 circulations were extended 7 minutes for back 68 ℃, with 1.5% agarose gel electrophoresis identification reaction product.Through identifying, from the total RNA of Catimor coffee berry, clone alpha-galactosidase cDNA coding region, obtained and expection about 1.2Kb segment of the same size.
The cDNA fragment of pcr amplification is connected to sequencing vector pGEMT-easy (Promega, 2800 Woods Hollow Road, Madison, WI 53711-5399, USA) in, according to method Transformed E .coli DH5 α (Sambrook etc., molecular cloning laboratory manual well-known in the art, second edition, press of cold spring harbor laboratory, cold spring port, New York, 1989), utilize plasmid purification test kit (Promega, 2800 Woods Hollow Road, Madison, WI 537u-5399 USA) carries out sequencing according to manufacturer's operational guidance extraction plasmid DNA.Utilize automatic dna sequencer to measure dna sequence dna, the result shows that what obtained is a kind of new isozyme of coffee berry alpha-galactosidase, promptly with clone's such as Zhu A. Santos coffee berry alpha-galactosidase cDNA sequence relatively, be C at the 798th Nucleotide, rather than T (the 233rd amino acids of encoding is constant, be Leu), at the 912nd Nucleotide is A, rather than G, is Thr thereby cause the 271st amino acids, rather than Ala, Yu Zhetong (Fig. 1).Independent cloning twice repeats sequencing result and all proves the sequence unanimity respectively.
The structure of Catimor coffee berry alpha-galactosidase recombinant expression vector pPICZ-Gal
Select the secreted expression carrier pPICZ α A (Invitrogen of thermophilic methanol yeast-pichia pastoris (Pichia pastoris) for use, 1600 Faraday Avenue, Carlsbad, CA 92008, USA) (Fig. 2 A), this carrier utilizes alcohol oxidase AOX1 promoter regulation goal gene to transcribe, and by the signal peptide mediation secreting, expressing of yeast saccharomyces cerevisiae α mating factor, the Zeocin resistant gene is as the screening sign.In multiple clone site, multidigit is named a person for a particular job and is made expressed exogenous genes products 5 ' end have some amino-acid residues of carrier sequence encoding, the XhoI site that has the signal peptide end only can guarantee that expression product is a natural form, but the encoding sequence (AAA AGA) that this site is positioned at signal peptide cutting site (Lys-Arg) before, therefore when construction of expression vector, complete for the restoring signal peptide, guarantee the consistence of reading frame, need add this two amino acid whose encoding sequences at Catimor coffee berry alpha-galactosidase coding sequence (signal peptide sequence that does not comprise himself) front end.In addition, for making things convenient for the purifying of expression product, the pPICZaA carrier comprises c-myc epi-position and poly Histidine metal-chelating district (6xHis), be positioned at foreign gene and insert the downstream, site, only need remove the termination codon of foreign gene self, can realize amalgamation and expression, design following primer sequence for this reason: upstream primer is 5 '-CCGCTCGAGAAAAGATTAGCAAATGGGCTT-3 '; Downstream primer is 5 '-GCTCTAGACACTGTGGGGTTAGGAC-3 '.With Hi-Fi Vent polysaccharase (NEW ENGLAND BIOLABS, 32 Tozer Road, Beverly, MA01915 USA) carries out pcr amplification, and reaction parameter is 94 ℃ of sex change 1 minute, annealed 2 minutes for 62 ℃, 72 ℃ were extended 3 minutes, and 35 circulations continue insulation extension 7 minutes for back 72 ℃.Amplified production is cut through the XhoI/XbaI enzyme, and agarose gel electrophoresis reclaims the 1.1kb fragment, inserts the XhoI/XbaI site of yeast vector pPICZaA, and the expression vector of structure is referred to as pPICZ-Gal (seeing Fig. 2 B).
Obtain the pichia pastoris recombinant bacterial strain of stably express alpha-galactosidase
Select single restriction enzyme site SacI in 5 ' AOX1 zone, alpha-galactosidase expression vector pPICZ-Gal (10 μ g) is carried out linearization for enzyme restriction, enzyme cuts full back (about 2 hours) and heats or add EDTA solution termination reaction, through phenol/chloroform extracting, ethanol sedimentation carries out sterilising treatment, last linearizing dna solution is (about 5～10 μ gDNA) in 10 μ l aseptic deionized waters, with according to (the 1600 Faraday Avenue of Invitrogen company, Carlsbad, CA92008,80 μ l pichia pastoris GS115 (Invitrogen of the specification sheets preparation that USA) provides, 1600 Faraday Avenue, Carlsbad, CA 92008, USA) competent cell mixes, forward in the aseptic electric shock cup of 0.2cm ice precooling, behind the ice bath 5 minutes, at 1500V, 25 μ F, shock by electricity under the 200 Ω conditions, the 1M sorbyl alcohol that adds the 1ml ice bath immediately, forward in the 15ml test tube, 30 ℃ of temperature are bathed 1～2 hour (not vibration), get 100～200 μ l solution coat on the YPD culture plate that contains microbiotic Zeocin (200 μ g/ml), 30 ℃ are incubated 2～4 days and occur until transformed clone.
Transformed clone is cultivated with methanol induction, follow the trail of the alpha-galactosidase expression by observing the colour-change that produces with the chromogenic substrate reaction, directly the screening positive expression is cloned: transformant is seeded on the nitrocellulose filter, be laid on the YPD flat board and cultivate, after treating that the clone grows up to, add 0.5% methyl alcohol, induce the alpha galactosides expression of enzymes, after 24 hours, film is placed on uses alpha-galactosidase chromogenic substrate 5-bromo-4-chloro-3-indoles-alpha galactosides (1mg/ml in advance, pH 6.5) on the wetted filter paper, room temperature is placed 15min, and the positive colony color will be gradually by xanthochromia indigo plant.Select the dozens of positive colony, be inoculated in the 5ml BMGY substratum, add 0.5% methanol induction and cultivate.Get the nutrient solution sample, the centrifugal 8min of 5000rpm collects supernatant, with supernatant and the single-minded substrate p-nitro-benzene of alpha-galactosidase base-α-D-galactopyranoside (1.25mmol/L, pH6.5) mix, 26 ℃ of incubations are after 1 hour, (0.1mol/L, pH9.8) termination reaction are measured 405nm place light absorption value to add sodium borate buffer liquid.The alpha-galactosidase activity of a unit (U) is defined as the alpha-galactosidase amount of per minute hydrolysis 1 μ mol substrate under above-mentioned testing conditions.Enzymic activity (U) calculation formula is:
[A
405nmX reacts cumulative volume (ml)]/[18.5X reaction times (branch)]
Relevant detailed content is referring to Kuo J-Y, Goldstein J, Enzyme MicrobiolTechnol 5:285,1983.
Detect the clone who wherein is numbered Z α-5 and can in every milliliter of culture supernatant, produce the nearly alpha-galactosidase activity of 12U.
Embodiment 4
The expression production of Catimor coffee berry alpha-galactosidase
Recombinant bacterial strain is 30 ℃ of enrichment and growth, 28 ℃ of abduction deliverings, the secreting, expressing amount that help reducing background albumen, improves target protein.The recombinant bacterial strain that is numbered Z α-5 that obtains is inoculated in 10ml BMGY nutrient solution (100mmol/L K
3PO
4, 1.34% YNB, 0.00004% vitamin H, 1% glycerine) in, 30 ℃ of shake-flask culture grow to cell density A
600nm=10～20, with centrifugal 8 minutes collecting cells of 5000rpm, be resuspended in 1/2 volume BMMY (100mmol/L K under the room temperature
3PO
4, 1.34% YNB, 0.00004% vitamin H, 0.5% methyl alcohol) in, 28 ℃ of abduction deliverings.Every 24 hours samples that take a morsel, and add methyl alcohol to final concentration 0.5%.According to the method for embodiment 3 sample determination specific activity to different time points, found that increase with induction time, expression product is accumulated, and the alpha galactosides specific enzyme activity raises gradually.Continuous induction was cultivated after 7 days, and detecting the supernatant total protein concentration is 1.2mg/ml, and enzymic activity reaches about 18U/ml (see figure 3).
The purifying of alpha-galactosidase expression product and determination of activity
Collect recombinant bacterial strain inducing culture supernatant, utilize Pharmacia KTA FPLC chromatographic system (Amersham Pharmacia, SE 751-84 Uppsala, Sweden), with metal chelate chromatography medium Chelating Sepharose FF separation, purifying alpha-galactosidase.The initial buffer system that purifying adopts is the 10mM imidazoles, 20mM NaPO
4, 0.5M NaCl (pH7.4-7.6) utilizes the 500mM imidazoles then, 20mM NaPO
4, 0.5M NaCl (pH7.4-7.6) buffer solution elution target protein.Behind the purifying, identify that through SDS-PAGE present single band, protein molecular weight is about the 41KD (see figure 4).Purified product is measured the alpha galactosides specific enzyme activity with preceding method, reaches 62U/mg, far above the level of Santos coffee berry alpha-galactosidase 30U/mg (Zhu A.1995, Arch.Biochem.Biophys, 324,65-70).
Embodiment 6
Catimor coffee berry alpha-galactosidase is used for B → O blood group to be changed
Substantially according to Goldstein J, Siviglia G, people such as Hurst R, science 215:168,1982 and Lenny L, Hurst R, people such as Goldstein J, Blood 77:1383-1388,1991 method is carried out.Gather the Type B red corpuscle of healthy blood donor,, wash cell 3 times, use isoosmotic phosphoric acid-citric acid-sodium-chlor damping fluid (PCS:60mM NaH again with physiological saline in 1250g centrifugation 4.8 minutes
2PO
4, the 25mM Trisodium Citrate, 75mM sodium-chlor pH5.5) is washed 4 times, and the PCS of pH5.6 washes 2 times, and every milliliter of Type B red corpuscle adds 50 or the alpha-galactosidase that obtains of the above-mentioned purifying of 100U, makes hematocrit value remain on 65-75%.26 ℃ of slow joltings, carry out enzyme digestion reaction.In the different time point sampling, the anti-A of standard that generally uses with clinical hospital, anti-B monoclonal antibody are (from Medical University Of Anhui, the blood grouping reagent that the southeast, Hefei medical science and technology Development Co., Ltd produces) carries out the agglutination reaction of serum test, observe at enzyme digestion reaction and carry out in the process, the aggregation of red corpuscle and anti-A antibody is without any variation, then weaken gradually with the aggregation of anti-B antibody, the 100U/ml reaction detected less than Type B antigen after 2 hours; Simultaneously, select a kind of of the said firm's production for use, make doubling dilution, detect more subtly and the erythrocytic aggregation of Type B by the different extension rates of 2-1024 for research anti-B monoclonal antibody that use, high titre.Experiment showed, under the reaction conditions identical with Santos coffee berry alpha-galactosidase (people such as Goldstein J, the source is the same; People such as Lenny L, the source is the same), use the high reactivity Catimor coffee berry alpha-galactosidase of 100U/ml, enzymolysis after 2 hours Type B red corpuscle and various anti-A, anti-B antibody the aggregation (see figure 5) does not all take place, present the feature of the general blood of O type, realize that successfully B → O blood group changes, and same reaction, Santos coffee berry alpha-galactosidase but need 180-200U (people such as GoldsteinJ, the source is the same; People such as Lenny L, the source is the same).
The The above results explanation, the specific activity of this novel isozyme significantly improves, and is used for B → O blood group and changes the consumption and the cost that can reduce enzyme greatly, for the practicability that B → O blood group changes is laid a good foundation.
Following biomaterial has been followed the regulation of Budapest treaty, is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is as follows:
Preservation thing preserving number preservation date pichia pastoris Z α-5 CGMCC No.0479 on July 21st, 2000
Described herein and claimed invention is not limited in the scope of disclosed specific embodiments, because these embodiments are intended to illustrate several aspect of the present invention.The embodiment of any equivalence includes within the scope of the invention.In fact, except shown in this paper and described, after the description of reference front, be conspicuous to those skilled in that art for modification of the present invention.These modifications are also contained in the scope of appended claims.When conflict is arranged,, comprise that definition is as the criterion with this paper disclosure.
This paper has quoted a lot of documents, and they all are incorporated herein by reference in full.
Sequence table＜110〉applicant: Feild Flood Transfusion Inst., Academy of Military Medicine Sciences, PLA＜120〉denomination of invention: polypeptide and code nucleic acid＜150 thereof with alpha-galactosidase activity〉application number: undetermined＜151〉applying date: undetermined＜160〉sequence number: 3＜210〉1＜211〉1207＜212〉DNA＜213〉fruitlet coffee Catimimor cultivars＜400〉11 GTTGGTGCTTCCGCT CGCCGGATGGTGAAG TCTCCAGGAACCGAG GATTACACTCGCAGG
MetValLys?SerProGlyThrGlu?AspTyrThrArgArg
1 5 1061 AGCCTTTTAGCAAAT?GGGCTTGGTCTAACA?CCTCCGATGGGGTGG?AACAGCTGGAATCAT
SerLeuLeuAlaAsn?GlyLeuGlyLeuThr?ProProMetGlyTrp?AsnSerTrpAsnHis
15 20 25 30121?TTCCGTTGTAATCTT?GATGAGAAATTGATC?AGGGAAACAGCCGAT?GCAATGGTATCAAAG
PheArgCysAsnLeu?AspGluLysLeuIle?ArgGluThrAlaAsp?AlaMetValSerLys
35 40 45 50181?GGGCTTGCTGCACTG?GGATATAAGTACATC?AATCTTGATGACTGT?TGGGCAGAACTTAAC
GlyLeuAlaAlaLeu?GlyTyrLysTyrIle?AsnLeuAspAspCys?TrpAlaGluLeuAsn
55 60 65 70241?AGAGATTCACAGGGG?AATTTGGTTCCCAAA?GGTTCAACATTCCCA?TCAGGGATCAAAGCC
ArgAspSerGlnGly?AsnLeuValProLys?GlySerThrPhePro?SerGlyIleLysAla
75 80 85 90301?TTAGCAGATTATGTT?CACAGCAAAGGCCTA?AAGCTTGGAATTTAC?TCTGATGCTGGAACT
LeuAlaAspTyrVal?HisSerLysGlyLeu?LysLeuGlyIleTyr?SerAspAlaGlyThr
95 100 105 110361?CAGACATGTAGTAAA?ACTATGCCAGGTTCA?TTAGGACACGAAGAA?CAAGATGCCAAAACC
GlnThrCysSerLys?ThrMetProGlySer?LeuGlyHisGluGlu?GlnAspAlaLysThr
115 120 125 130?421?TTTGCTTCATGGGGG?GTAGATTACTTAAAG?TATGACAACTGTAAC?AACAACAACATAAGC
PheAlaSerTrpGly?ValAspTyrLeuLys?TyrAspAsnCysAsn?AsnAsnAsnIleSer
135 140 145 150?481?CCCAAGGAAAGGTAT?CCAATCATGAGTAAA?GCATTGTTGAACTCT?GGAAGGTCCATATTT
ProLysGluArgTyr?ProIleMetSerLys?AlaLeuLeuAsnSer?GlyArgSerIlePhe
155 160 165 170?541?TTCTCTCTATGTGAA?TGGGGAGAGGAAGAT?CCAGCAACATGGGCA?AAAGAAGTTGGAAAC
PheSerLeuCysGlu?TrpGlyGluGluAsp?ProAlaThrTrpAla?LysGluValGlyAsn
175 180 185 190?601?AGTTGGAGAACCACT?GGAGATATAGATGAC?AGTTGGAGTAGCATG?ACTTCTCGGGCAGAT
SerTrpArgThrThr?GlyAspIleAspAsp?SerTrpSerSerMet?ThrSerArgAlaAsp
195 200 205 210?661?ATGAACGACAAATGG?GCATCTTATGCTGGT?CCCGGTGGATGGAAT?GATCCAGACATGCTG
MetAsnAspLysTrp?AlaSerTyrAlaGly?ProGlyGlyTrpAsn?AspProAspMet
215 220 225 230?721?GAGGTGGGAAATGGA?GGCATGACTACAACG?GAATATCGATCCCAT?TTCAGCATTTGGGCA
GluValGlyAsnGly?GlyMetThrThrThr?GluTyrArgSerHis?PheSerIleTrpAla
235 240 245 250?781?TTAGCAAAAGCACCT?CTACTGATTGGCTGT?GACATTCGATCCATG?GACGGTACGACTTTC
LeuAlaLysAlaPro?LeuLeuIleGlyCys?AspIleArgSerMet?AspGly
ThrPhe
255 260 265 270?841?CAACTGCTAAGCAAT?GCGGAAGTTATTGCG?GTTAACCAAGATAAA?CTTGGCGTTCAAGGG
GlnLeuLeuSerAsn?AlaGluValIleAla?ValAsnGlnAspLys?LeuGlyValGlnGly
275 280 285 290?901?AACAAGGTTAAGACT?TACGGAGATTTGGAG?GTTTGGGCTGGACCT?CTTAGTGGAAAGAGA
AsnLysValLysThr?TyrGlyAspLeuGlu?ValTrpAlaGlyPro?LeuSerGlyLysArg
295 300 305 310?961?GTAGCTGTCGCTTTG?TGGAATAGAGGATCT?TCCACGGCTACTATT?ACCGCGTATTGGTCC
ValAlaValAlaLeu?TrpAsnArgGlySer?SerThrAlaThrIle?ThrAlaTyrTrpSer
315 320 325 3301021?GACGTAGGCCTCCCG?TCCACGGCAGTGGTT?AATGCACGAGACTTA?TGGGCGCATTCAACC
AspValGlyLeuPro?SerThrAlaValVal?AsnAlaArgAspLeu?TrpAlaHisSerThr
335 340 345 3501081?GAAAAATCAGTCAAA?GGACAAATCTCAGCT?GCAGTAGATGCCCAC?GATTCGAAAATGTAT
GluLysSerValLys?GlyGlnIleSerAla?AlaValAspAlaHis?AspSerLysMetTyr
355 360 365 3701141?GTCCTAACCCCACAG?TGATTAACAGGAGAA?TGCAGAA
ValLeuThrProGln?***
375 378＜210〉2＜211〉378＜212〉protein＜213〉fruitlet coffee Catimor Cultivars＜400〉2MetValLysSerPro GlyThrGluAspTyr ThrArgArgSerLeu LeuAlaAsnGlyLeu, 15 10 15 20GlyLeuThrProPro MetGlyTrpAsnSer TrpAsnHisPheArg CysAsnLeuAspGlu
25 30 35 40LysLeuIleArgGlu?ThrAlaAspAlaMet?ValSerLysGlyLeu?AlaAlaLeuGlyTyr
45 50 55 60LysTyrIleAsnLeu?AspAspCysTrpAla?GluLeuAsnArgAsp?SerGlnGlyAsnLeu
65 70 75 80ValProLysGlySer?ThrPheProSerGly?IleLysAlaLeuAla?AspTyrValHisSer
85 90 95 100LysGlyLeuLysLeu?GlyIleTyrSerAsp?AlaGlyThrGlnThr?CysSerLysThrMet
105 110 115 120ProGlySerLeuGly?HisGluGluGlnAsp?AlaLysThrPheAla?SerTrpGlyValAsp
125 130 135 140TyrLeuLysTyrAsp?AsnCysAsnAsnAsn?AsnIleSerProLys?GluArgTyrProIle
145 150 155 160MetSerLysAlaLeu?LeuAsnSerGlyArg?SerIlePhePheSer?LeuCysGluTrpGly
165 170 175 180GluGluAspProAla?ThrTrpAlaLysGlu?ValGlyAsnSerTrp?ArgThrThrGlyAsp
185 190 195 200IleAspAspSerTrp?SerSerMetThrSer?ArgAlaAspMetAsn?AspLysTrpAlaSer
205 210 215 220TyrAlaGlyProGly?GlyTrpAsnAspPro?AspMet
GluVal?GlyAsnGlyGlyMet
225 230 235 240ThrThrThrGluTyr?ArgSerHisPheSer?IleTrpAlaLeuAla?LysAlaProLeuLeu
245 250 255 260IleGlyCysAspIle?ArgSerMetAspGly
ThrPheGlnLeu?LeuSerAsnAlaGlu
265 270 275 280ValIleAlaValAsn?GlnAspLysLeuGly?ValGlnGlyAsnLys?ValLysThrTyrGly
285 290 295 300AspLeuGluValTrp?AlaGlyProLeuSer?GlyLysArgValAla?ValAlaLeuTrpAsn
305 310 315 320ArgGlySerSerThr?AlaThrIleThrAla?TyrTrpSerAspVal?GlyLeuProSerThr
325 330 335 340AlaValValAsnAla?ArgAspLeuTrpAla?HisSerThrGluLys?SerValLysGlyGln
345 350 355 360IleSerAlaAlaVal?AspAlaHisAspSer?LysMetTyrValLeu?ThrProGln***
365 370 375 378＜210〉fruit coffee Bourbon Santos cultivar＜400〉3MetValLysSerPro GlyThrGluAspTyr ThrArgArgSerLeu LeuAlaAsnGlyLeu, 15 10 15 20GlyLeuThrProPro MetGlyTrpAsnSer TrpAsnHisPheArg CysAsnLeuAspGlu 3＜211〉378＜212〉protein＜213 〉
25 30 35 40LysLeuIleArgGlu?ThrAlaAspAlaMet?ValSerLysGlyLeu?AlaAlaLeuGlyTyr
45 50 55 60LysTyrIleAsnLeu?AspAspCysTrpAla?GluLeuAsnArgAsp?SerGlnGlyAsnLeu
65 70 75 80ValProLysGlySer?ThrPheProSerGly?IleLysAlaLeuAla?AspTyrValHisSer
85 90 95 100LysGlyLeuLysLeu?GlyIleTyrSerAsp?AlaGlyThrGlnThr?CysSerLysThrMet
105 110 115 120ProGlySerLeuGly?HisGluGluGlnAsp?AlaLysThrPheAla?SerTrpGlyValAsp
125 130 135 140TyrLeuLysTyrAsp?AsnCysAsnAsnAsn?AsnIleSerProLys?GluArgTyrProIle
145 150 155 160MetSerLysAlaLeu?LeuAsnSerGlyArg?SerIlePhePheSer?LeuCysGluTrpGly
165 170 175 180GluGluAspProAla?ThrTrpAlaLysGlu?ValGlyAsnSerTrp?ArgThrThrGlyAsp
185 190 195 200IleAspAspSerTrp?SerSerMetThrSer?ArgAlaAspMetAsn?AspLysTrpAlaSer
205 210 215 220TyrAlaGlyProGly?GlyTrpAsnAspPro?AspMet
GluVal?GlyAsnGlyGlyMet
225 230 235 240ThrThrThrGluTyr?ArgSerHisPheSer?IleTrpAlaLeuAla?LysAlaProLeuLeu
245 250 255 260IleGlyCysAspIle?ArgSerMetAspGly?AlaThrPheGlnLeu?LeuSerAsnAlaGlu
265 270 275 280ValIleAlaValAsn?GlnAspLysLeuGly?ValGlnGlyAsnLys?ValLysThrTyrGly
285 290 295 300AspLeuGluValTrp?AlaGlyProLeuSer?GlyLysArgValAla?ValAlaLeuTrpAsn
305 310 315 320ArgGlySerSerThr?AlaThrIleThrAla?TyrTrpSerAspVal?GlyLeuProSerThr
325 330 335 340AlaValValAsnAla?ArgAspLeuTrpAla?HisSerThrGluLys?SerValLysGlyGln
345 350 355 360IleSerAlaAlaVal?AspAlaHisAspSer?LysMetTyrValLeu?ThrProGln***
365 370 375 378
Claims (30)
1. the isolated polypeptide with alpha-galactosidase activity is characterized in that
(1) described polypeptide has the aminoacid sequence identical with aminoacid sequence shown in the SEQ ID NO:2 at least 80%,
(2) alpha-galactosidase activity of this polypeptide is higher than having shown in the SEQ ID NO:3 more than the sequence alpha-galactosidase activity of peptide at least.
2. the polypeptide of claim 1, wherein said polypeptide have the aminoacid sequence identical with aminoacid sequence shown in the SEQ ID NO:2 at least 90%.
3. the polypeptide of claim 1, wherein said polypeptide have the aminoacid sequence identical with aminoacid sequence shown in the SEQ ID NO:2 at least 95%.
4. the polypeptide of claim 1, wherein said polypeptide comprises the aminoacid sequence of SEQ ID NO:2.
5. the polypeptide of claim 1, its aminoacid sequence or its subsequence by SEQ ID NO:2 is formed.
6. the polypeptide of claim 1, it can derive from fruitlet coffee.
7. the polypeptide of claim 1, it can derive from fruitlet coffee Catimor Cultivar.
8. the polypeptide of any one among the claim 1-7, the alpha-galactosidase activity of this polypeptide are at least than having shown in the SEQ ID NO:3 alpha-galactosidase activity of peptide high about 25% more than the sequence.
9. the polypeptide of claim 8, the alpha-galactosidase activity of this polypeptide are at least than having shown in the SEQ ID NO:3 alpha-galactosidase activity of peptide high about 50% more than the sequence.
10. the polypeptide of claim 8, the alpha-galactosidase activity of this polypeptide are higher by 75% than having shown in the SEQ ID NO:3 more than the sequence alpha-galactosidase activity of peptide at least.
11. the polypeptide of claim 8, the alpha-galactosidase activity of this polypeptide is higher at least by about 100% than having shown in the SEQID NO:3 more than the sequence alpha-galactosidase activity of peptide.
12. polypeptide that comprises aminoacid sequence shown in the SEQ ID NO:2.
13. the isolated polypeptide with alpha-galactosidase activity is characterized in that:
(1) described polypeptide has the aminoacid sequence identical with aminoacid sequence shown in the SEQ ID NO:2 at least 80%, and
(2) with this polypeptide and p-nitro-benzene base-α-D-galactopyranoside 1.25mmol/L, pH6.5 mixes, and 26 ℃ of incubations are after 1 hour, and the alpha-galactosidase activity that records is higher than 30U/mg at least.
14. the polypeptide of claim 13, the alpha-galactosidase activity that it is characterized in that this polypeptide is at least about the described pure polypeptide of 40U/mg.
15. the polypeptide of claim 13, the alpha-galactosidase activity that it is characterized in that this polypeptide is at least about 50U/mg.
16. the polypeptide of claim 13, the alpha-galactosidase activity that it is characterized in that this polypeptide is at least about 62U/mg.
17. comprise the nucleotide sequence of coding claim 1 polypeptide and/or the isolating polynucleotide of its complementary sequence.
18. be included in the peptide coding nucleotide sequence of SEQ ID NO:1 the nucleotide sequence of at least one sudden change and/or the isolating polynucleotide of its complementary sequence are arranged, wherein said mutant nucleic acid sequence encoding contains the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.
19. comprise the polynucleotide of claim 17 or 18, and the nucleic acid construct that can be operatively connected, can instruct one or more control sequence that polypeptide produces with it in suitable expressive host.
20. a recombinant expression vector wherein comprises the nucleic acid construct of claim 19.
21. a recombinant host cell wherein comprises the nucleic acid construct of claim 19.
22. according to the recombinant host cell of claim 21, it is a pichia pastoris.
23. according to the recombinant host cell of claim 21, it is CGMCC No.0479.
24. the method for the polypeptide of production claim 1 or 13 comprises this polypeptide of purifying from fruitlet coffee.
25. the method for claim 24, wherein said fruitlet coffee is the Catimor Cultivar.
26. the method for each polypeptide among the production claim 1-16 comprises that (a) is being suitable for producing under the condition of this polypeptide, cultivates the host cell that contains nucleic acid construct, this nucleic acid construct comprises the nucleotide sequence of this polypeptide of encoding; (b) reclaim this polypeptide.
27. the polypeptide of producing by the method for claim 24 or 25.
28. the polypeptide of producing by the method for claim 26.
29. the application of the polypeptide of any one in B → O blood group conversion among the claim 1-16,27 and 28.
30. B → O blood group conversion method, this method may further comprise the steps:
(1) separate the Type B hemocyte,
(2) with suitable isotonic buffer solution washing Type B hemocyte,,
(3) add any one described polypeptide among the claim 1-16,
(4) at 20-26 ℃ of incubation one to a few hours,
(5) use the phosphate buffered saline(PBS) washed cell.
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| CNB00122736XA Expired - Fee Related CN1197872C (en) | 2000-08-11 | 2000-08-11 | Polypeptide with alpha-galactosidase activity and its coding nucleic acid |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100389836C (en) * | 2004-12-10 | 2008-05-28 | 北京市红十字血液中心 | Tetrabag used for B-O blood type transition closed treatment and its use method |
| CN100398655C (en) * | 2005-10-11 | 2008-07-02 | 山东大学 | Transglycosyl alpha-galactoglucosidezyme gene |
| CN100526458C (en) * | 2006-05-16 | 2009-08-12 | 中国农业科学院饲料研究所 | Alpha-galactosidase gene, its coding protein, production and use |
| CN102154411A (en) * | 2011-01-07 | 2011-08-17 | 山东大学 | Method for preparing Globotriose oligosaccharide |
| CN101712930B (en) * | 2008-10-08 | 2011-09-21 | 广东溢多利生物科技股份有限公司 | Novel antiprotease acid alpha-galactosidase AGA36 and gene and application thereof |
| CN102321599A (en) * | 2011-10-25 | 2012-01-18 | 云南师范大学 | Low-temperature alpha-galactosidase AgaAGN14 and gene thereof |
| CN107267486A (en) * | 2014-09-29 | 2017-10-20 | 中国农业科学院生物技术研究所 | A kind of beta galactosidase combination mutant with high transglycosylation and its preparation method and application |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101892207B (en) * | 2010-07-23 | 2011-12-28 | 中国农业科学院饲料研究所 | Low-temperature alpha-galactosidase GalA17, gene thereof and application thereof |
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- 2000-08-11 CN CNB00122736XA patent/CN1197872C/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100389836C (en) * | 2004-12-10 | 2008-05-28 | 北京市红十字血液中心 | Tetrabag used for B-O blood type transition closed treatment and its use method |
| CN100398655C (en) * | 2005-10-11 | 2008-07-02 | 山东大学 | Transglycosyl alpha-galactoglucosidezyme gene |
| CN100526458C (en) * | 2006-05-16 | 2009-08-12 | 中国农业科学院饲料研究所 | Alpha-galactosidase gene, its coding protein, production and use |
| CN101712930B (en) * | 2008-10-08 | 2011-09-21 | 广东溢多利生物科技股份有限公司 | Novel antiprotease acid alpha-galactosidase AGA36 and gene and application thereof |
| CN102154411A (en) * | 2011-01-07 | 2011-08-17 | 山东大学 | Method for preparing Globotriose oligosaccharide |
| CN102321599A (en) * | 2011-10-25 | 2012-01-18 | 云南师范大学 | Low-temperature alpha-galactosidase AgaAGN14 and gene thereof |
| CN102321599B (en) * | 2011-10-25 | 2013-01-09 | 云南师范大学 | Low-temperature alpha-galactosidase AgaAGN14 and gene thereof |
| CN107267486A (en) * | 2014-09-29 | 2017-10-20 | 中国农业科学院生物技术研究所 | A kind of beta galactosidase combination mutant with high transglycosylation and its preparation method and application |
| CN107267486B (en) * | 2014-09-29 | 2020-05-12 | 中国农业科学院生物技术研究所 | β -galactosidase combined mutant with high transglycosidic activity and preparation method and application thereof |
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|---|---|
| CN1197872C (en) | 2005-04-20 |
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