CN1261103A - Human targeting glycogen protein,and its coding sequence, preparing process and application - Google Patents

Abstract

The present invention invention discloses a new human protein, it is The Human Protein Targeting to Glycogen (HPTG), which is the homolog of mouse PTG and human PP1R5. The polynucleotide sequence for coding the protein polypeptide, the application of said polynucleotide and polypeptide and the process for preparing said polynucleotide and said polypeptide are also disclosed.

Description

Human Protein Targeting to Glycogen and encoding sequence thereof, and method for making and purposes

The present invention relates to the genetically engineered field, particularly, the present invention relates to a kind of new human gene nucleotide sequence.More particularly, the present invention relates to the cDNA sequence of Human Protein Targeting to Glycogen (Human Protein Targeting toG1ycogen abbreviates " HPTG " as), this albumen is the homologue of mouse PTG.The invention still further relates to by this nucleotide sequence coded polypeptide the application of these polynucleotide and polypeptide, and the production method of described polynucleotide and described polypeptide.

A large amount of evidences show that proteinic reversible phosphorylation is being regulated all many-sides of cell function.The effect of phosphoprotein phosphatase and protein kinase makes the phosphorylation process reversible.Phosphoprotein phosphatase-1 (PP1) is a class serine-threonine phosphatase, and it is present in all eukaryotic cells, participates in the regulation and control of many cell functions, comprises metabolism, Muscle contraction, mitotic the stopping of glycogen

The albumen that one class is called as guiding subunit (being also referred to as binding subunit) plays an important role in phosphoprotein phosphatase cell regulate and control process.The diversity of phosphoprotein phosphatase and substrate thereof shows the strict regulation and control of enzymic activity needs.The bootable phosphoprotein phosphatase of guiding subunit is to specific ubcellular site, regulate substrate characteristic and catalysis characteristics, and make this enzyme pair cell external signal make correct response, and the fidelity of assurance phosphoprotein phosphatase (Trends.Biochem.Sci.1993,18:172-177).

A kind of binding subunit G _MBootable PP1 is to striate glycogenosome and sarcoplasmic reticulum, G _MThe Ser-46 phosphorylation can increase the dephosphorylation speed of PP1, thereby activate glycogen synthetase.In contrast, the phosphorylation of Ser-45 can suppress PP1, glycogen is synthesized suppressed, thereby stimulate glycogen regeneration.Another kind of binding subunit G _LBootable PP1 combines the dephosphorylation speed that the back increases PP1 to liver starch with PP1, thereby activates glycogen synthetase.They have common to participate in and PP1 bonded motif: RVXF, and this motif exists also in other many binding subunits that (EMBO J.1997; 16 (8): 1876-1887; Adv.Prot.Phosphatases 1996; 8:371-376).G _LAnd G _MBe found (Eur.J.Biochem.1996 with PP1 bonded structural domain in the binding subunit; 239:317-325).

In Mammals, also found the binding subunit of many other PP1, comprising the unstriated muscle myosin in conjunction with the guiding mixture (by M ₁₁₀And M ₂₁Form) (Eur.J.Biochem.1992; 210:1023-1035), p53 conjugated protein (53BP2) (FEBS Lett.1995; 377:295-300), nucleoprotein NIPP-1 (J.Biol.Chem.1995; 270; 28068-28074), RNA splicing factor PST1 (FEBS Lett.1996; 389:183-189).Report some other albumen and PP1 reaction in addition, for example retinoblastoma product (Genes Dev.1993; 7:555-569), ribosomal protein L 5 (J.Biol.Chem.1995; 270:19786-19790) etc.

Recently, find the PP1 binding subunit of another 36.4kDa in mouse, it is called as targeting glycogen protein (PTG, protein targeting to glycogen), and (Science 1997; 275:1475-1478), this albumen is called as PPP1R5 (Protein phosphatase 1 binding subunit R5, protein phosphatase 1 binding subunit R5) (FEBS Lett.1996 in the people; 399:339-343).This albumen can to activate glycogen not synthetic relying under the situation of hormone, and wide spectrum expresses, and is subjected to the regulation and control of Phosphoric acid esterase not obvious yet.

Studies show that further the enhancing of PTG is expressed can influence Glycogen Metabolism.Experimental result shows that the overexpression meeting of PTG in mouse liver cell obviously activates the activity of glycogen synthetase, and it is synthetic to activate glycogen, does not even depend on glucose and Regular Insulin; The overexpression of PTG also can stop the glycogenolysis of cAMP mediation.These results show that the overexpression of PTG is locked in glycogenesis pattern (J.Biol.Chem.1998 to cell; 273 (41): 26421-26425).Experiment also shows, in the Chinese hamster ovary cell of overexpression insulin receptor, the overexpression of PTG can obviously increase the synthetic and insulin stimulating glycogen down of basic glycogen and synthesize, this prompting PTG plays an important role in Glycogen Metabolism, may glycogen synthetase, Starch phosphorylase α, phosphorylating kinase and PP1 be assemblied on the glycogenosome as a kind of minute submounts, form the metabolism complex body, (Science 1997 to accept intracellular signal on the spot; 275:1475-1478).

An object of the present invention is to provide a kind of new polynucleotide, a newcomer of this polynucleotide encoding phosphoprotein phosphatase binding subunit family, phosphoprotein phosphatase binding subunit family member of the present invention is named as HPTG.

Another object of the present invention provides a kind of new human protein phosphatase enzyme binding subunit family member, and this albumen is named as HPTG albumen.

A further object of the present invention provides a kind of method of utilizing recombinant technology to produce described new people HPTG polypeptide.

The present invention also provides the application of this people HPTG nucleotide sequence and polypeptide.

In one aspect of the invention, a kind of isolated dna molecular is provided, it comprises: coding has the nucleotide sequence of the polypeptide of people HPTG protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 10-960 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 10-960 position.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO.4.More preferably, this sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 10-960 position.

In another aspect of this invention, provide a kind of isolating people HPTG protein polypeptide, it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.4 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.4 polypeptide of sequence.

In another aspect of this invention, provide a kind of carrier, it contains above-mentioned isolated DNA.

In another aspect of this invention, provide a kind of described carrier transformed host cells.

In another aspect of this invention, provide a kind of generation to have the method for the polypeptide of people HPTG protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people HPTG protein-active operationally is connected in expression regulation sequence, form people HPTG protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 10-960 position among described nucleotide sequence and the SEQ ID NO.3;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of people HPTG;

(c) under the condition that is fit to expressing human HPTG protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people HPTG protein-active.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 965 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 10-960 position Nucleotide.

In the present invention, " isolating ", " purifying " or " pure substantially " DNA are meant, this DNA or fragment have been arranged in the sequence of its both sides and have separated under native state, refer to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separate with the protein of in cell, following it.

In the present invention, term " people HPTG albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with people HPTG protein-active is as 10-960 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 10-960 position Nucleotide of SEQ ID NO.3 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID NO.3 in 10-960 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.4 of also encoding out.This term also comprises can be under the moderate stringent condition, more preferably under the height stringent condition, with among the SEQ ID NO.3 from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 10-960 position.Also term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 10-960 position, preferably at least 80%, at least 90% nucleotide sequence more preferably.

This term also comprises encoding to have variant form with proteic, the SEQ ID NO.4 sequence of people HPTG identical function.These variant forms comprise (but being not limited to): several (are generally 1-90, preferably 1-60, more preferably 1-20,1-10 best) disappearance, insertion and/or the replacement of Nucleotide, and several (are generally in 60 to hold interpolation 5 ' and/or 3 ', preferably being in 30, more preferably is in 10, is in 5 best) Nucleotide.

In the present invention, " pure substantially " protein or polypeptide are meant that it accounts at least 20% of the total material of sample at least, preferably at least 50%, more preferably at least 80%, and at least 90% (by dry weight or weight in wet base) best.Purity can be measured with any suitable method, as measure the purity of polypeptide with column chromatography, PAGE or HPLC method.Substantially pure polypeptide is substantially free of the component of following it under the native state.

In the present invention, term " people HPTG protein polypeptide " refers to have the SEQ IDNO.4 polypeptide of sequence of people HPTG protein-active.This term also comprises having and variant form people HPTG identical function, SEQ ID NO.4 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of people HPTG and reactive derivative.

Polypeptide variant form of the present invention comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of people HPTG DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people HPTG polypeptide to obtain.The present invention also provides other polypeptide, as comprises people HPTG polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people HPTG polypeptide.Usually, this fragment have people HPTG peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.

Invention also provides the analogue of people HPTG albumen or polypeptide.The difference of these analogues and natural human HPTG polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.

In the present invention, " people HPTG conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID No.4, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.

Table 1

Initial residue	Representational replacement	The preferred replacement
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe；	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

The present invention also comprises people HPTG polypeptid coding sequence and segmental antisense sequences thereof.This antisense sequences can be used for suppressing the expression of people HPTG in the cell.

The present invention also comprises a kind of nucleic acid molecule that can be used as probe, and this molecule has 8-100 of people HPTG polypeptid coding sequence, preferably 15-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample nucleic acid molecule of coding people HPTG.

The present invention also comprises the method that detects people HPTG nucleotide sequence, and it comprises with above-mentioned probe and sample and hybridizing whether detection probes combination has taken place then.Preferably, this sample is the product behind the pcr amplification, and wherein the pcr amplification primer is corresponding to the encoding sequence of people HPTG polypeptide, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 20-50 Nucleotide.

In the present invention, can select various carrier known in the art for use, as commercially available carrier.Such as, select commercially available carrier for use, the nucleotide sequence with code book invention polypeptide operationally is connected in expression regulation sequence then, can form protein expression vector.

As used herein, " operationally being connected in " refers to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjoining, then means in reading frame adjacent for the secretion leader sequence.

In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.Eukaryotic host cell commonly used comprises yeast cell, insect cell and mammalian cell.Preferably, this host cell is an eukaryotic cell, as Chinese hamster ovary celI, COS cell etc.

On the other hand, the present invention also comprises people HPTG DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people HPTG gene product or fragment.Preferably, refer to that those can combine with people HPTG gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of people HPTG, comprise that also those do not influence the antibody of people HPTG protein function.The present invention also comprise those can with modify or without the people HPTG gene product bonded antibody of modified forms.

The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab) ₂Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule (people such as Ladner, U.S. Patent No. 4,946,778); Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.

Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the people HPTG gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human HPTG or its have antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare that (see people such as Kohler, Nature 256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People .In Monoclonal Antibodies and T Cell Hybridomas such as Hammerling, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block people HPTG function and the antibody that does not influence people HPTG function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people HPTG gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of people HPTG gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).

People HPTG Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.

In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.

In one embodiment of the invention, the cDNA nucleotide sequence of HPTG is so to obtain, with human brain λ gt11cDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer---A1:5 '-CTTTGCCTAATGAGCTGCACCAG-3 ' is a forward primer, oligonucleotide A2:5 '-TTAATTCACGAAAAGGGCCAGTTC-3 ' is a reverse primer, carries out PCR.Obtain the full length cDNA sequence of SEQID NO.3 after the order-checking.

Studies show that, in Mammals with PP1 bonded subunit in, (Science 1997 for targeting glycogen protein (PTG, proteintargeting to glycogen); 275:1475-1478) or PPP1R5 (FEBS Lett.1996; 399:339-343) can to activate glycogen not synthetic relying under the situation of hormone, and wide spectrum expresses, and is subjected to the regulation and control of Phosphoric acid esterase also not obvious.The enhancing of PTG is expressed can influence Glycogen Metabolism.The overexpression of PTG in rat hepatocytes can obviously activate the activity of glycogen synthetase and activate glycogen and synthesize, and can stop the glycogenolysis of cAMP mediation in addition.The overexpression of PTG is locked in glycogenesis pattern (J.Biol.Chem.1998 to cell; 273 (41): 26421-26425).Experiment also shows, in the Chinese hamster ovary cell of overexpression insulin receptor, the overexpression of PTG can obviously increase the basis synthetic (Science 1997 with the glycogen of insulin stimulating; 275:1475-1478).Therefore, the PTG proteinoid plays an important role in Glycogen Metabolism.

In the accompanying drawings,

Fig. 1 is the homology comparison diagram of the aminoacid sequence of people HPTG of the present invention and people PP1R5.Wherein, identical amino acid marks with " | " between two sequences, and similar amino acid marks with " ".Similar amino acid is: A, S, T; D, E; N, Q; R, K; I, L, M, V; F, Y, W.

Fig. 2 is the homology comparison diagram of the aminoacid sequence of people HPTG of the present invention and mouse PTG.Wherein, identical amino acid marks with " | " between two sequences, and similar amino acid marks with " ".Similar amino acid is: A, S, T; D, E; N, Q; R, K; I, L, M, V; F, Y, W.

Fig. 3 is 3 kinds of proteic amino acid sequence homologous comparison diagrams between inventor HPTG and people PP1R5 and the mouse PTG.Wherein, identical amino acid marks with " * " below sequence, and similar amino acid marks with " ".

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The clone and the mensuration of the cDNA sequence of people HPTG

1. primer amplification

With human brain λ gt11cDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer---A1:5 '-CTTTGCCTAATGAGCTGCACCAG-3 ' (SEQ ID NO:1) is a forward primer, oligonucleotide A2:5 '-TTAATTCACGAAAAGGGCCAGTTC-3 ' (SEQ ID NO:2) is a reverse primer, carries out PCR.The PCR condition of A1/A2 be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 66 ℃ 1 minute and 70 ℃ thereupon, last 70 ℃ were extended 5 minutes.Electrophoresis detection obtains the purpose fragment of about 1kb.

2.PCR the order-checking of product

Above-mentioned pcr amplification product A1/A2 is connected with pGEM-T carrier (Promega), transformed into escherichia coli JM103, extract plasmid with QIAprep Plasmid test kit (QIAGEN), carry out the mistake of orientations Lieque with double-stranded nested type disappearance test kit (Pharmacia) to inserting fragment, carry out Rapid identification and ordering with PCR to lacking son then.Use SequiTherm EXCEL ^TMDna sequencing kit (Epicentre Technologies) checks order to disappearance of brachymemma successively, with computer software splicing order, obtain full length cDNA sequence at last, altogether 965bp, detailed sequence is seen SEQ ID NO:3, and wherein open reading frame is positioned at 10-960 position Nucleotide.

Derive the aminoacid sequence of people HPTG according to the complete encoding sequence that obtains, totally 316 amino-acid residues, its aminoacid sequence sees SEQ ID NO:4 for details.

Embodiment 2

Homology relatively

Full length cDNA sequence and proteins encoded thereof with people HPTG of the present invention, in Non-redundantGenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDStranslations+PDB+SwissProt+Spupdate+PIR database, carry out nucleic acid and albumen homology retrieval with blast program.Found that it and people PPP1R5 gene and proteins encoded thereof have homology, the identity on protein level reaches 93%, and similarity is 94% (Fig. 1).In addition, have higher homology with the PTG of mouse, identity is 81%, and similarity is 86% (Fig. 2).

To amino acid conserved regions analysis revealed, there is motif RVXF (in a Fig. 3 underscore part relevant with the PP1 keying action in people HPTG albumen of the present invention with its homologue on protein structure, wherein X represents arbitrary amino acid), therefore, people HPTG albumen of the present invention can be included into the conjugated protein family of PP1.

Phosphoprotein phosphatase-1 (PP1) is the class in the serine-threonine phosphatase, is present in all eukaryotic cells, participates in the regulation and control of many cell functions.The albumen that one class is called as guiding subunit (being also referred to as binding subunit) plays an important role in phosphoprotein phosphatase cell regulate and control process.This class subunit can arrive specific ubcellular site by the pilot protein Phosphoric acid esterase, regulate substrate characteristic and catalysis characteristics, and make this enzyme pair cell external signal make correct response, and the fidelity of assurance phosphoprotein phosphatase (Trends.Biochem.Sci.1993,18:172-177).

Studies show that further the enhancing of PTG is expressed can influence Glycogen Metabolism.Experiment shows, in the Chinese hamster ovary cell of overexpression insulin receptor, the overexpression of PTG can obviously increase the synthetic and insulin stimulating glycogen down of basic glycogen and synthesize, this prompting PTG plays an important role in Glycogen Metabolism, may glycogen synthetase, Starch phosphorylase α, phosphorylating kinase and PP1 be assemblied on the glycogenosome as a kind of minute submounts, form the metabolism complex body, (Science 1997 to accept intracellular signal on the spot; 275:1475-1478).Experiment also result shows that the overexpression of PTG in mouse liver cell obviously activates the activity of glycogen synthetase, and it is synthetic to activate glycogen, does not even depend on glucose and Regular Insulin; The overexpression of PTG stops the glycogenolysis of cAMP mediation, these results show, the overexpression of PTG is locked in glycogenesis pattern to cell, prompting PTG can be used for improving the deposit of liver cell glucose, may be used for the treatment of the hypoglycemia (J.Biol.Chem.1998 that the diabetes that do not rely on Regular Insulin cause; 273 (41): 26421-26425).

People HPTG of the present invention is used for further functional study except can be used as this family's a member, also can be used for producing fusion rotein with other albumen, such as producing fusion rotein with immunoglobulin (Ig).In addition, inventor HPTG can also merge with other members of this family or exchange fragment, to produce new albumen.For example the N end of inventor HPTG and the N end of mouse PTG are exchanged, to produce the albumen that new activity is higher or have new features.

At the antibody of inventor HPTG, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family).

In addition, inventor HPTG nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people HPTG or the overexpression that suppresses people HPTG.People HPTG albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people HPTG disappearance, no function or unusual cause related disorders arranged.High homology prompting by HPTG of the present invention and people PPP1R5 and mouse PTG, the present invention may be as the former metabolism of submounts involved in sugar in a kind of minute, to some hormone, the information response that transmits as Regular Insulin, thus certain effect may when not relying on the hypoglycemia that the diabetes of Regular Insulin cause, be arranged in treatment.

In addition, can also be with carrying out relevant diagnosis or prognosis judgement based on nucleotide sequence of the present invention or antibody.

Embodiment 3

The expression of people HPTG in intestinal bacteria

In this embodiment, with the cDNA sequence (the pcr amplification product A1/A2 of embodiment 1) of coding people HPTG use corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers of 3 ' end increase, obtain people HPTG cDNA as inserting fragment.

5 ' Oligonucleolide primers sequence of using in the PCR reaction is:

5′-TCAGGGATCC?ATGAGCTGCA?CCAGAATGAT-3′(SEQ?ID?NO.5)，

This primer contains the restriction enzyme site of BamHI restriction enzyme, is 20 Nucleotide of the people HPTG encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5’-TTGCAAGCTT?TCACGAAAAG?GGCCAGTTC-3’(SEQ?ID?NO.6)，

This primer contains the part encoding sequence of restriction enzyme site, translation termination and the people HPTG of HindIII restriction enzyme.

The restriction enzyme site of the restriction enzyme on the primer is corresponding to bacterial expression vector pQE-9 (Qiagen Inc., Chatsworth, the CA) restriction enzyme digestion sites on, this plasmid vector coding antibiotics resistance (Amp ^r), a bacterium replication orgin (ori), an adjustable promotor/operon of IPTG-(P/O), a ribosome bind site (RBS), a 6-histidine mark thing (6-His) and restriction enzyme cloning site.

With SalI and HindIII digestion pQE-9 carrier and insertion fragment, will insert fragment subsequently and be connected to the pQE-9 carrier and keep open reading frame initial at bacterium RBS.Transform available from Qiagen with connecting mixture subsequently, the E.coli bacterial strain of commodity M15/rep4 by name, M15/rep4 contains the plasmid pREP4 of multiple copied, and it is expressed the lacI repressor and carries kalamycin resistance (Kan ^r).Screen transformant containing on the LB culture dish of Amp and Kan, the extracting plasmid, the cDNA fragment of sequence verification people HPTG has correctly been inserted carrier.

Incubated overnight (O/N) contains the positive transformant clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml) and Kan (25 μ g/ml).Spend the night (O/N) culture with 1: 100-1: 250 thinning ratio dilution, be inoculated into then in the large volume substratum, culturing cell grows to 600 optical density(OD) (OD ₆₀₀) when being 0.4-0.6, add IPTG (" isopropylthio-") to final concentration be 1mM.By making lacI repressor inactivation, IPTG induces startup P/O to cause gene expression dose to improve.Continued culturing cell 3-4 hour, centrifugal subsequently (6000 * g, 20 minutes).The ultrasonic degradation inclusion body, collecting cell also is dissolved in cell precipitation in the Guanidinium hydrochloride of 6M.After the clarification, by containing under the condition that 6-His marker albumen combines closely making, with nickel-chelate column chromatography purifying dissolved people HPTG from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out people HPTG from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.Perhaps, use the dialysis step to remove Guanidinium hydrochloride, perhaps isolated purifying protein from nickel-chelate column.Protein behind the purifying is incorporated in second post, has the linear Guanidinium hydrochloride gradient of successively decreasing in this post.Protein denaturation when being attached to this post is used Guanidinium hydrochloride (pH5.0) wash-out subsequently.At last, soluble protein is dialysed with PBS, then protein is kept in the stock solution that final concentration is 10% (w/v) glycerine.

SDS-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is about 36KDa.

In addition, the amino acid that reaches proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.4 with ordinary method.

Embodiment 4

The expression of people HPTG in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, with the cDNA sequence (the pcr amplification product A1/A2 of embodiment 1) of coding people HPTG use corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers of 3 ' end increase, obtain people HPTG cDNA as inserting fragment.

5 ' Oligonucleolide primers sequence of using in the PCR reaction is:

5′-TCAGAAGCTT?ATGAGCTGCA?CCAGAATGAT-3′(SEQ?ID?NO.7)

This primer contains the restriction enzyme site of HindIII restriction enzyme, is 20 Nucleotide of the people HPTG encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5’-TTGCGGATCC?TCACGAAAAG?GGCCAGTTC-3’(SEQ?ID?NO.8)

This primer contains the part encoding sequence of the restriction enzyme site of BamHI restriction enzyme, translation termination and people HPTG.

The restriction enzyme site of the restriction enzyme on the primer is corresponding to the restriction enzyme digestion sites on the expressing cho cell carrier pcDNA3, this plasmid vector coding antibiotics resistance (Amp ^rAnd Neo ^r), a phage replication starting point (f1 ori), a virus replication starting point (SV40 ori), T7 promotor, a viral promotors (P-CMV), a Sp6 promotor, a SV40 promotor, a SV40 tailing signal and corresponding polyA order, a BGH tailing signal and a corresponding polyA order.

With HindIII and EcoRI digestion pcDNA3 carrier and insertion fragment, will insert fragment subsequently and be connected to the pcDNA3 carrier.Subsequently with connecting mixture Transformed E .coli DH5 α bacterial strain.Screen transformant containing on the LB culture dish of Amp, incubated overnight (O/N) contains the clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml).Extracting plasmid, PstI enzyme are cut and are identified insertion clip size and direction, and the cDNA fragment of sequence verification people HPTG has correctly been inserted carrier.

The plasmid transfection Chinese hamster ovary celI is to adopt lipofection, carries out with Lipofectin (GiBco Life).After the transfection 48 hours, through the lasting G418 pressurization screening in 2-3 week, collecting cell and cell conditioned medium are measured the expressing protein enzyme activity.Remove G418, continuous passage is cultivated; To mixing the clone cell Method of Limited Dilution, select to have the cell subclone of higher protein-active.The above-mentioned positive subclone of a large amount of according to a conventional method cultivations.After 48 hours, beginning collecting cell and supernatant are with ultrasonic degradation method smudge cells.With 50mMTris-HCl (pH7.6) solution that contains 0.05%Triton is balance liquid and elutriant, uses through the Superdex of pre-equilibration G-75 post and collects above-mentioned proteic active peak.Using 50mMTris-HCl (pH8.0) equilibrated DEAE-Sepharose post again, is that elutriant carries out gradient elution with 50mMTris-HCl (pH8.0) solution that contains 0-1M NaCl, collects above-mentioned proteic active peak.Be that dialyzate is dialysed to expressing protein solution with PBS (pH7.4) then.Last freeze-drying is preserved.

SDS-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is 36kDa.

In addition, the amino acid that reaches proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.4 with ordinary method.

Embodiment 5

Preparation antibody

The recombinant protein that obtains in embodiment 3 and 4 is used for immune animal to produce antibody, and concrete grammar is as follows.Recombinant molecule is standby after separating with chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days,, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml with the same antigen of non-complete Freund ' s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least three times.The sero-fast specific reaction that obtains is active to be assessed in the ability of external precipitation people HPTG gene translation product with it.Found that antibody can precipitate with protein-specific of the present invention ground specifically.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table (1) general information:

(i) applicant: Fudan University

(ii) denomination of invention: Human Protein Targeting to Glycogen and encoding sequence thereof, and method for making and purposes

(iii) sequence number: the information of 8 (2) SEQ ID NO.1

(i) sequence signature

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: the information of SEQ ID NO.1:AGATTACTAT GGAGATCTGG CTG 23 (2) SEQ ID NO.2

(i) sequence signature

(A) length: 24 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: the information of SEQ ID NO:2TTAATTCACG AAAAGGGCCA GTTC 24 (2) SEQ ID NO.3:

(i) sequence signature:

(A) length: 965bp

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: cDNA

( iii ) ：SEQ ID NO.3CTTTGCCTAA TGAGCTGCAC CAGAATGATC CAGGTTTTAG ATCCACGTCC TTTGACAAGT 60TCGGTCATGC CCGTGGATGT GGCCATGAGG CTTTGCTTGG CACATTCACC ACCTGTAAAG 120AGTTTCCTGG GCCCGTACGA TGAATTTCAA CGACGACATT TTGTGAATAA ATTAAAGCCC 180CTGAAATCAT GTCTCAATAT AAAACACAAA GCCAAATCAC AGAATGACTG GAAGTGCTCA 240CACAACCAAG CCAAGAAGCG CGTTGTGTTT GCTGACTCCA AGGGCCTCTC TCTCACTGCG 300ATCCATGTCT TCTCCGACCT CCCAGAAGAA CCAGCGTGGG ATCTGCAGTT TGATCTCTTG 360GACCTTAATG ATATCTCCTC TGCCTTAAAA CACCACGAGG AGAAAAACTT GATTTTAGAT 420TTCCCTCAGC CTTCAACCGA TTACTTAAGT TTCCGGAGCC ACTTTCAGAA GAACTTTGTC 480TGTCTGGAGA ACTGCTCGTT GCAAGAGCGA ACAGTGACAG GGACTGTTAA AGTCAAAAAT 540GTGAGTTTTG AGAAGAAAGT TCAGATCCGT ATCACTTTCG ATTCTTGGAA AAACTACACT 600GACGTAGACT GTGTCTATAT GAAAAATGTG TATGGTGGCA CAGATAGTGA TACCTTCTCA 660TTTGCCATTG ACTTACCCCC TGTCATTCCA ACTGAGCAGA AAATTGAGTT CTGCATTTCT 720TACCATGCTA ATGGGAAGTC TTTCGGGGAC AACAATGATG GTCAGAATTA TTGGATTGTT 780CATGTTCAAT GGAAGCCTGA TGGGGTGCAG ACACAGGTGG AACCCCAGGA CTGTGAATTC 840CACCAGACGC CTTCTAAGAC AGAGATAGGG TCAACAATCT TTGGCAGGCC AAGGCTTGCT 900TACGGGCTCT CCCCAGAGTG GAAAGGTTTG GGGGGAGTTG AGAACTGGCC CTTTTCGTGA 960ATTAA ( 2 ) SEQ ID NO.4：

(i) sequence signature:

(A) length: 316 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: polypeptide

( xi ) ：SEQ ID NO.4Met Ser Cys Thr Arg Met Ile Gln Val Leu Asp Pro Arg Pro Leu 15Thr Ser Ser Val Met Pro Val Asp Val Ala Met Arg Leu Cys Leu 30Ala His Ser Pro Pro Val Lys Ser Phe Leu Gly Pro Tyr Asp Glu 45Phe Gln Arg Arg His Phe Val Asn Lys Leu Lys Pro Leu Lys Ser 60Cys Leu Asn Ile Lys His Lys Ala Lys Ser Gln Asn Asp Trp Lys 75Cys Ser His Asn Gln Ala Lys Lys Arg Val Val Phe Ala Asp Ser 90Lys Gly Leu Ser Leu Thr Ala Ile His Val Phe Ser Asp Leu Pro 105Glu Glu Pro Ala Trp Asp Leu Gln Phe Asp Leu Leu Asp Leu Asn 120Asp Ile Ser Ser Ala Leu Lys His His Glu Glu Lys Asn Leu Ile 135Leu Asp Phe Pro Gln Pro Ser Thr Asp Tyr Leu Ser Phe Arg Ser 150His Phe Gln Lys Asn Phe Val Cys Leu Glu Asn Cys Ser Leu Gln 165Glu Arg Thr Val Thr Gly Thr Val Lys Val Lys Asn Val Ser Phe 180Glu Lys Lys Val Gln Ile Arg Ile Thr Phe Asp Ser Trp Lys Asn 195Tyr Thr Asp Val Asp Cys Val Tyr Met Lys Asn Val Tyr Gly Gly 210Thr Asp Ser Asp Thr Phe Ser Phe Ala Ile Asp Leu Pro Pro Val 225Ile Pro Thr Glu Gln Lys Ile Glu Phe Cys Ile Ser Tyr His Ala 240Asn Gly Lys Ser Phe Gly Asp Asn Asn Asp Gly Gln Asn Tyr Trp 255Ile Val His Val Gln Trp Lys Pro Asp Gly Val Gln Thr Gln Val 270Glu Pro Gln Asp Cys Glu Phe His Gln Thr Pro Ser Lys Thr Glu 285Ile Gly Ser Thr Ile Phe Gly Arg Pro Arg Leu Ala Tyr Gly Leu 300Ser Pro Glu Trp Lys Gly Leu Gly Gly Val Glu Asn Trp Pro Phe 315Ser ( 2 ) SEQ ID NO.5

(i) sequence signature

(A) length: 30 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: the information of SEQ ID NO.5:TCAGGGATCC ATGAGCTGCA CCAGAATGAT 30 (2) SEQ ID NO.6

(i) sequence signature

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi sequence description: the information of SEQ ID NO.6:TTGCAAGCTT TCACGAAAAG GGCCAGTTC 29 (2) SEQ ID NO.7

(i) sequence signature

(A) length: 30 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: the information of SEQ ID NO.7:TCAGAAGCTT ATGAGCTGCA CCAGAATGAT 30 (2) SEQ ID NO.8

(i) sequence signature

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO.8:TTGCGGATCC TCACGAAAAG GGCCAGTTC 29

Claims (14)

1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the polypeptide of people HPTG protein-active,

Show at least 70% homology from the nucleotides sequence of Nucleotide 10-960 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps

Described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 10-960 position.

2. dna molecular as claimed in claim 1 is characterized in that, described sequence encoding one polypeptide, and this polypeptide has the sequence shown in the SEQ ID NO.4.

3. dna molecular as claimed in claim 1 is characterized in that, this sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 10-960 position.

4. isolating people HPTG protein polypeptide is characterized in that it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.4 aminoacid sequence, or its reactive derivative.

5. polypeptide as claimed in claim 4 is characterized in that, this polypeptide is to have SEQ ID NO.4 polypeptide of sequence.

6. a carrier is characterized in that, it contains the described DNA of claim 1.

7. one kind with the described carrier transformed host cells of claim 6.

8. host cell as claimed in claim 7 is characterized in that this cell is intestinal bacteria.

9. host cell as claimed in claim 7 is characterized in that this cell is an eukaryotic cell.

10. a generation has the method for the polypeptide of people HPTG protein-active, it is characterized in that this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people HPTG protein-active operationally is connected in expression regulation sequence, form people HPTG protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 10-960 position among described nucleotide sequence and the SEQ ID NO.3;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of people HPTG;

(c) under the condition that is fit to expressing human HPTG protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people HPTG protein-active.

11. method as claimed in claim 10 is characterized in that, this sequence is from Nucleotide 10-960 position among the SEQ ID NO.3.

12. energy and the described people HPTG of claim 4 protein polypeptide specificity bonded antibody.

13. a nucleic acid molecule is characterized in that, it is the antisense sequences of the described dna molecular of claim 1.

14. a probe molecule is characterized in that, it contains 8-100 continuous nucleotide in the described dna molecular of claim 1.

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