CN1596304A - Protein knobs - Google Patents
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- CN1596304A CN1596304A CNA02822101XA CN02822101A CN1596304A CN 1596304 A CN1596304 A CN 1596304A CN A02822101X A CNA02822101X A CN A02822101XA CN 02822101 A CN02822101 A CN 02822101A CN 1596304 A CN1596304 A CN 1596304A
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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Abstract
Compositions containing a knob attached to a specific site of a protein and methods of producing and using these compositions are disclosed. The compositions comprise a knob, a tail portion and a protein portion. The protein portion contains a substituted cysteine residue at the desired location of labeling. The tail portion is located at the terminal end of the protein portion. The knob is linked to the end of the tail portion and contains a cysteine residue. The substituted cysteine residue on the protein portion and the cysteine residue on the knob form a disulfide bond, thereby tagging the protein portion at the desired site with the knob.
Description
Government's rights and interests
This research is subjected to the subsidy of approval number NICHD HD14907 of NIH and NICHDHD38547.The present invention finishes under the subsidy of United States Government.Government can have some right of the present invention.
Invention field
The present invention relates to the protein labeling field.
Foreword
Analysing protein interphase interaction at present or the used protein labeling method of protein purification comprise marker and proteinic C-terminal or N-terminal fusion, or insert residue in proteinic rings.These methods all have certain limitation.For example, no matter be or use protein that and only molecule is invested protein terminal and compare, carrying out mark at proteinic specific position may be desirable more with dye molecule.These comprise be designed for detection as hCG and its acceptor protein and macromolecular substance between Study of Interaction.In addition, when proteinic end participated in this proteinic function, carrying out mark at proteinic end may be not ideal enough.The method that marker is inserted in the protein loop also has limitation, and this is because if marker does not insert between the protein domain, then marker usually necessary smaller (several residue number).Sometimes different big or small probes being attached to protein surface may be even more ideal.In addition, use cysteine residues to modify also very difficulty, because the halfcystine that protein may contain other cysteine residues that need protection or be sealed, protein may sex change after removing the sealing residue.Therefore, the application of labelled protein in industry of these methods generations is restricted.
For instance because the complex construction of human chorionic gonadotrophin (hCG) and with metakentrin acceptor (LHR) in the interactional similarity in a plurality of sites, be difficult to identify this hormone and the contacted position of LHR acceptor.The chain that the crystalline structure of human chorionic gonadotrophin (hCG) shows its β subunit as " securing band (seatbelt) " around α subunit (Lapthorn, A.J., Harris, D.C., Littlejohn, A., Lustbader, J.W., Canfield, R.E., Machin, K.J., Morgan, F.J.﹠amp; Isaacs, N.W. (1994) Nature 369,455-461; Wu, H., Lustbader, J.W., Liu, Y., Canfield, R.E.﹠amp; Hendrickson, W.A. (1994) Structure 2,545-558.).As if most of protein dimer is kept stable fully by being in contact with one another between subunit, and hCG is different therewith, and it is mainly fixed by its securing band; If the disulfide linkage of Cys26 in " pinning (latches) " securing band C-terminal and the β subunit core is removed, then may be by making unstable hCG secretion (Suganuma, N., Matzuk, the M.M.﹠amp of destroying of heterodimer; Boime, I. (1989) J.Biol.Chem.264,19302-19307).The evolution sexual clorminance of this unusual structural rearrangement is still unclear, and it may reflect viewed phenomenon when some hCG analogues combine with fsh receptor---allow subunit in the heterodimer be moved (Wang, Y.H., Bernard, M.P.﹠amp; Moyle, W.R. (2000) Mol.Cell.Endocrinol.170,67-77.).
Comprise hCG all three class sugar-protein hormones acceptor all with the coupling of G protein, and have and contain a plurality of big ectodomain (Segaloff, D.L.﹠amp that are rich in the leucine tumor-necrosis factor glycoproteins; Ascoli, M. (1993) Endocr.Rev.14,324-347.).Back one finds that the prompting ectodomain may to be rich in leucine repeat sequence protein part similar to other, is Horseshoe (Kobe, B.﹠amp; Deisenhofer, J. (1993) Nature 366,751-756).As if two zones of ectodomain relevant with its part bonded avidity and specificity.HCG is similar with the avidity to complete acceptor to the avidity of the LHR analogue of alternative montage and brachymemma, the amino-acid residue at prompter LHR ectodomain N-terminal 2/3rds places has formed the binding site (Braun of high-affinity, T., Schofield, P.R.﹠amp; Sprengel, R. (1991) EMBO.J.10,1885-1890; Thomas, D., Rozell, T.G., Liu, X.﹠amp; Segaloff, D.L (1996) Mol.Endocrinol.10,760-768.), the amino-acid residue at ectodomain C-terminal 1/5th places disturbs itself and the combining of non-human mammal metakentrin, this part contact (Bernard that in fact is mainly on the space with contacting of acceptor that this hormone is pointed out in this discovery, M.P., Myers, R.V.﹠amp; Moyle, W.R. (1998) Biochem.J.335,611-617.).
Still there is dispute in the surface that hCG, hFSH and hTSH most possibly contact with LHR, HSHR and THSHR.α subunit C-terminal (Wu, H., Lustbader, J.W., Liu, Y., Canfield, R.E.﹠amp adjacent in the heterodimer have been found with the part securing band; Hendrickson, W.A. (1994) Structure 2 545-558.) influences avidity (Lapthorn, A.J., Harris, the D.C. of all sugar-protein hormones to its acceptor, Littlejohn, A., Lustbader, J.W., Canfield, R.E., Machin, K.J., Morgan, F.J.﹠amp; Isaacs, N.W. (1994) Nature 369,455-461; Bernard, M.P., Myers, R.V.﹠amp; Moyle, W.R. (1998) Biochem.J.335,611-617), and just to be proposed as in the past in more than 25 years be acceptor contact (Lapthorn, A.J., Harris, D.C., Littlejohn, A., Lustbader, J.W., Canfield, R.E., Machin, K.J., Morgan, F.J.﹠amp; Isaacs, N.W. (1994) Nature 369,455-461.).The 26S Proteasome Structure and Function data of this hormone are lumped together consideration, and these discoveries cause the hormone different with ultimate principle-receptor complex viewpoint (Moyle, W.R., Campbell, R.K., Rao, S.N.V., Ayad, N.G., Bernard, M.P., Han, Y.﹠amp; Wang, Y. (1995) J.Biol.Chem.270,20020-20031).These viewpoints contact (Jiang, X., Dreano, M., Buckler, D.R., Cheng, S., Ythier, A., Wu, H., Hendrickson, W.A., Tayar, N.E.﹠amp from this hormone with the concave surface of acceptor ectodomain; El Tayar, N. (1995) Structure 3 is 1341-1353) to hormone and acceptor edge contact (Moyle, W.R., Campbell, R.K., Rao, S.N.V., Ayad, N.G., Bernard, M.P., Han, Y.﹠amp; Wang, Y. (1995) J.Biol.Chem.270 20020-20031) has.Second ring of all viewpoint prompting α subunits of hCG-LHR mixture has part in the face of acceptor, still has to be determined but whether this part of hormone participates in the acceptor contact.Activity (Peng, K.C., Bousfield, G.R., Puett, D.﹠amp that this ring sudden change can the reduction of report explanation hCG is arranged; Ward, D.N. (1996) Journal of Protein Chemistry 15,547-552; Xia, H., Chen, F.﹠amp; Puett, D. (1994) Endocrinol.134 1768-1770), points out it may facilitate essential LHR contact.
Protein-acceptor interaction is to understand the function of cell and the key of behavior regulation and control thereof.Many work, as design new medicine, all need deeply understand protein-acceptor interaction.But, still have many restrictions understanding protein-acceptor interaction at present.Be familiar with the 26S Proteasome Structure and Function of this protein conformation, and how this proteinic structure is favourable with acceptor interaction (this receptor also is protein usually).If a certain proteinic other aspects are known, proteinic certain aspect can be inferred by knowledge and experience sometimes.Protein-acceptor interaction can be simulated on computers, but simulation is the task of a complexity, especially because molecule is deformable, and takes the similar conformation of big energy.The simulation of protein-its receptor is also very difficult, because need considering to be subjected to stops, the characteristic of part and solvent.
Except that simulation, another approach of understanding protein-acceptor interaction is to change this protein structure and receptor structure and test condition in being provided with, and measures changing or the effect of disappearance in conjunction with the effect that changes and to protein function.These experimental techniques also have the limitation part, because handle this protein or acceptor has certain difficulty, the ability of measuring these changes is also limited.Therefore will be a much progress in this area by the invention that continues to cause or arrestin matter-acceptor interaction improves the experimental data tolerance range, because it can measure its function that causes or afunction.
In principle, utilize site-directed mutagenesis technique should be easy to illustrate the part that hormone and acceptor are in contact with one another.Unfortunately, the sudden change of α subunit C-terminal and β subunit securing band can change position (Jiang, X., Dreano, M., the Buckler of subunit in heterodimer, D.R., Cheng, S., Ythier, A., Wu, H., Hendrickson, W.A., Tayar, N.E.﹠amp; El Tayar, N. (1995) Structure 3,1341-1353; Pierce, J.G.﹠amp; Parsons, T.F. (1981) Annu.Rev.Biochem.50 465-495), therefore is difficult to explain the influence of these key components of this hormone to its function.And sudden change may change crucial LHR contact, mobile subunit position or the two haves both at the same time, and the hCG activity is changed.Really, to the hormone-receptor interaction big influential sudden change of α subunit C-terminal (Pierce, J.G.﹠amp; Parsons, T.F. (1981) Annu.Rev.Biochem.50,465-495, Chen, F., Wang, Y.﹠amp; Puett, D. (1992) Mol.Endocrinol.6 is 914-919) with securing band sudden change (Campobell, R.K., DeanEmig, D.M.﹠amp; Moyle, W.R. (1991) Proc.Natl.Acad.Sci. (USA) 88,760-764; Campbell, R.K., Bergert, E.R., Wang, Y., Morris, J.C.﹠amp; Moyle, W.R. (1997) Nature Biotech.15,439-443; Grossmann, M., Szkudlinski, M.W., Wong, R., Dial, J.A., Ji, T.H.﹠amp; Weintraub, B.D. (1997) J.Biol.Chem.272,15532-15540; Lindau-Shepard, B., Roth, K.E.﹠amp; Dias, J.A. (1994) Endocrinol.135 1235-1240) also can change position (Wang, Y.H., Bernard, the M.P.﹠amp of subunit in heterodimer; Moyle, W.R. (2000) Mol.Cell.Endocrinol.170,67-77; Moyle, W.R., Campbell, R.K., Rao, S.N.V., Ayad, N.G., Bernard, M.P., Han, Y.﹠amp; Wang, Y. (1995) J.Biol.Chem.270,20020-20031).Differentiate in the protein and comprise the residue that discriminating does not contact with acceptor with another alternative method of acceptor interaction part.These residues can more sure being identified.Yet, obtain to be used to differentiate that the probe of these residues has certain difficulty.Correspondingly, the interactional probe production method of analysing protein also needs to improve.
Interact except being used for analysing protein, on probe or the molecule specific position in addition the protein of mark powerful tool is provided.The at present used method that probe is connected or is marked on molecular end is restricted their use but as previously mentioned.Other method comprises complicated program, needs various reactions and blocking group not to change the structure of other amino acid and this functional protein to generate pointed decoration.Correspondingly, the fixed point marking method of improvement also is essential.
Protein purification usually is necessary but some heavy step.The protein of purifying may be the necessary intermediate product of a certain scientific experimentation, also may be dead end product.Lipidated protein is to the success of test and treatment normally crucial.In some cases, injection protein must be removed any pollutent or be proved that it is harmless by the examination of food and medicine Surveillance Authority.Except that purity, protein must keep its biological activity.
Although all methods all have certain limitation, many known protein matter purification process are arranged in the art.The size exclusion chromatography helps separation substantially, but is not a kind of method of strictness, and need be with sample concentration.Gel electrophoresis can be come out the protein clean cut separation in the mixture, but only to the small sample practicality.Affinity chromatography is a useful method, but need filter out parent pollutant usually.Accordingly, can help to use the proteinic invention of minority step clean cut separation will be that one in this area big marked improvement, especially this invention can guarantee purity and when enlarging the purifying capacity.
Except these purposes, tubercle (knob) can be used for " covering " proteinic particular surface.This class purposes can be used in the prodrug in design, and these prodrugs can be used for leading tumour or other unnecessary tissues suffer from the unnecessary tissue of finding in the SF ovary of polycystic ovarian syndrome as diagnosis.Like this, tubercle is connected near the reactive site of toxin or toxicity enzyme, the toxicity enzyme can be used in the patient.In case this toxin or enzyme arrive a certain position, the contained enzyme in this position can cut and be connected with nodular connector, and the activity of this toxin or enzyme is recovered.This strategy is estimated to reduce unnecessary side effect and is taken place, otherwise these side effects may limit the application of this toxin or enzyme.Equally, tubercle can be used for blocking the activity of reagent such as the PTEN that promotes apoptosis.
Summary of the invention
The present invention relates to comprise proteinic composition, preparation of compositions and the using method thereof of specific site (site) with the tubercle mark.Tubercle refers to and can be customized to be used for the protein label of specific end use.
In one aspect of the present invention, disclose at the protein of specific site with the tubercle mark.The protein of specific site mark comprises tubercle, afterbody and protein portion.Tubercle comprises marker or probe feature (aspect), and has cysteine residues.Afterbody is between tuberal part branch and protein portion.Protein portion has in the need mark and has substituted original amino acid whose cysteine residues.The cysteine residues of nodular cysteine residues and protein portion forms disulfide linkage.In the present invention on the other hand, afterbody can comprise proteolytic enzyme or other cleavage sites.
In the another aspect of the present invention, the method for producing protein with tubercle fixed point mark is disclosed.These methods comprise protein that need to select mark, are needing selection marquee site on the protein of mark, generate desired protein; Wherein need the site of mark to substitute in the desired protein with halfcystine is residual, desired protein has at one end also comprised afterbody simultaneously, and contain nodosity at the end of afterbody, wherein tubercle contains cysteine residues, and cysteine residues in the tubercle and the cysteine residues in the protein portion form disulfide linkage.In related fields of the present invention, the method that tubercle is connected to the hCG specific site is disclosed.These methods comprise that can express the construct transfered cell that natural hCG β or hCG β-S138C, natural hCG α or hCG α-halfcystine substitute analogue makes it coexpression, and with 140 or 145 residues fusions of tubercle and hCG β.
In the another aspect of the present invention, the protein that discloses with site-specific nature mark among the present invention comes method for purifying proteins.These methods comprise in certain proteinic construct transfered cell of coding, wherein coded protein is included in the site that needs mark and carries out the alternate cysteine residues, be positioned at protein one end, have the afterbody of cysteine residues and cleavage site, and the tubercle part that is positioned at tail end; The condition that construct is expressed is provided; Lysing cell and according to the feature protein purification of protein superior thyroid tubercle.
In the another aspect of the present invention, the nodular using method of protein of pointed decoration is disclosed.For example, the protein tubercle can be used for the distance between mapping (map) protein, survey the surface of protein-protein contact surface, between two incoherent protein, form mixture, survey the proteinic 26S Proteasome Structure and Function of protein tubercle, proteinaceous solid due to the surface, is transported to cell with protein as target protein, also can be used for protein purification.Those of skill in the art will recognize in this area, be the strong research tool with extensive use with the custom indicia thing at the protein of specific site mark.
The accompanying drawing summary
The 3-D view of Fig. 1, hCG.Demonstration can be by the hCG α subunit residue of hCG β subunit C-terminal scanning (scan).The skeleton of α subunit and β subunit is represented with black and grayish band respectively.The β afterbody is represented with black stripe.The position that C alpha-carbon atom in the effectively crosslinked alternative cysteine residues takes place between α subunit residue and probe halfcystine is represented with black ball.More shallow grey ball refers to produce the less residue of crosslinked amount.Greyish white bead refers to that the insignificant halfcystine of crosslinked amount substitutes.Annotate: as if to such an extent as to the motor capacity of the 90th, 91 and 92 residue of α subunit can not find them too by force in the crystalline structure of hCG, these residues approximate location shown here only is in order to emphasize that they obviously have the ability of pinning securing band.
The combination of Fig. 2 A, hCG or hCG analogue, wherein a natural residue in second ring of analogue α subunit is substituted by halfcystine.
The cAMP of Fig. 2 B, hCG or hCG analogue gathers, and wherein a natural residue in second ring of analogue α subunit is substituted by halfcystine.
Fig. 3 A, the influence of α subunit C-terminal halfcystine alternate.The combination of hCG or hCG analogue, wherein a residue of analogue α subunit C-terminal is substituted by halfcystine.
Fig. 3 B, the influence of α subunit C-terminal halfcystine alternate.The cAMP of hCG or hCG analogue gathers, and wherein a residue of analogue α subunit C-terminal is substituted by halfcystine.
The combination of Fig. 4 A, hCG or hCG analogue, wherein analogue contains the β afterbody that is connected to second ring of α subunit.
The cAMP of Fig. 4 B, hCG or hCG analogue gathers, and wherein analogue contains the α afterbody that is connected to second ring of α subunit.
The combination of Fig. 5 A, hCG or hCG analogue, wherein analogue contains the β afterbody that is connected to α subunit carboxyl terminal residue.
The cAMP of Fig. 5 B, hCG or hCG analogue gathers, and wherein analogue contains the β afterbody that is connected to α subunit carboxyl terminal residue.
The combination of Fig. 6, analogue and signaling activity, wherein the α subunit residue of analogue is connected with BLA.
Fig. 7, α subunit and contain the aminoacid sequence of the mutant of alternate halfcystine.(annotate: suddenly change with capitalization and highlighted demonstration.These mutant prepare with cassette mutagenesis conventional in this area and PCR mutafacient system.)(SEQ?ID?NO：1～SEQ?ID?NO：35)。
The aminoacid sequence of Fig. 8, β subunit analogs.(annotate: substitute halfcystine with capitalization and highlighted demonstration.)(SEQ?ID?NO：36～SEQ?ID?NO：42)。
Fig. 9 A, protein tubercle, wherein tubercle is connected in the end of protein C-terminal.
Fig. 9 B, protein tubercle, wherein tubercle is connected in the end of protein amino end.
Figure 10 A, protein tubercle, wherein tubercle comprises cysteine residues.
Figure 10 B, protein tubercle, wherein tubercle comprises the aminoacid sequence that contains cysteine residues, merges with protein.
Figure 10 C, protein tubercle, wherein nodular cysteine residues is positioned at the proteinic surface of this tubercle.
Figure 11, tubercle are to the active influence of FSH.
Crosslinked chimeric analogs was summed up with respect to the activity of CF101-109 during Figure 12, LHR and FSHR measured, and wherein CF101-109 is a kind of difunctional mosaic that does not contain hCG β subunit C-terminal.
The influence that Figure 13, tubercle conduct the fsh receptor signal.
The influence that Figure 14 A, tubercle conduct the LH receptor signal.
The influence that Figure 14 B, tubercle conduct the LH receptor signal.
The aminoacid sequence of Figure 15, other analogues (SEQ ID NO:43～SEQ ID NO:53).
The signal conduction and the combination of Figure 16, long heterodimer and short heterodimer are active.
Figure 17, contain the metakentrin activity of the nodular hCG analogue of β-Nei Xiananmei.
The cAMP of the analogue of Figure 18, hCG and disappearance α Asn52 oligosaccharides gathers.
Figure 19, α K44A+hCG β combine with LHR's.
Figure 20 A, hCG and hCG analogue α K44E, K45Q+hCG β combine with LHR's.
Figure 20 B, reply the LHR cAMP of hCG and α K44E, K45Q+hCG β.
Figure 21 A, hCG and hCG analogue α K91E+hCG β combine with LHR's.
Figure 21 B, LHR cAMP gather the relative reactivity of hCG and α K91E+hCG β in the analysis.
Figure 22 A, reply the LHR cAMP of hCG and α K91M+hCG β.
Figure 22 B, hCG and α K91M+hCG β combine with LHR's.
Figure 23, hCG and contain the analogue of brachymemma connector and combining of LHR.
Figure 24, hCG and contain the analogue of brachymemma connector and combining of LHR.
Figure 25, hCG and α N52C+hCG β, S138C is to the hormesis of LHR cAMP.
Figure 26, analogue combine with LHR's, and the C-terminal of the α subunit of analogue adds a tubercle with the hCG β subunit carboxyl afterbody of brachymemma on 96,97 or 99 residues of hCG β subunit.
The LHR signal conduction of Figure 27, analogue, the C-terminal of the α subunit of analogue adds a tubercle with the hCG β subunit C-terminal of brachymemma on 98 or 99 residues of hCG β subunit.
Figure 28, analogue combine with LHR's, and the hCG β subunit carboxyl afterbody with brachymemma in the analogue adds a tubercle on 95 or 96 residues of β subunit.
The LHR signal conduction of Figure 29, analogue, the hCG β subunit carboxyl afterbody with brachymemma in the analogue adds a tubercle on 95 or 96 residues of hCG β subunit.
Figure 30 with the analogue of disappearance afterbody and be added with a nodular analogue with the hCG β subunit carboxyl afterbody of brachymemma on 96 residues of hCG/hFSH mosaic β subunit and compare, the LHR signal that is added with a nodular analogue with the GGC tail on 96 residues of β subunit conducts.
The LHR signal conduction of Figure 31, analogue, the hCG β subunit carboxyl afterbody with brachymemma in the analogue adds a tubercle on the 98th or 99 residue of β subunit.
The LHR signal conduction of Figure 31, analogue has shown that afterbody to tubercle being added to the influence of the 95th residue of hCG β subunit, reaches not having the influence of the analogue that makes 52 glycosylated abilities of residue of α subunit.
Figure 32, the cyclisation AMP that crosslinked analogue is replied.
Detailed Description Of The Invention
At present, most of labelled protein all is the end that marker is connected to proteinic N-terminal or C-terminal, or the minority residue is inserted protein loop, is prepared from the form of fused protein.Although the end mark thing can be any size, the mark that inserts in the protein loop is confined to the less relatively amino-acid residue of number usually, and marker inserts except the situation between the protein domain.Can introduce halfcystine in the position of need mark, the mercapto groups with halfcystine reacts with the sulfydryl specific reagent then, thus in different positions to protein mark in addition.But, when preparing under the condition that this protein comprises other halfcystines or causing halfcystine to be closed, finish very difficulty of preparation, and the situation that the latter often meets when to be protein express in eukaryotic cell.
The invention provides improving one's methods of labelled protein.Method of the present invention has avoided introducing as mentioned above the relevant complicated factor that halfcystine comes mark, makes the probe or the tubercle of different sizes also can be connected to proteinic surface except being connected to proteinic end.The invention provides the method that " tubercle " of different sizes is connected to hCG surface specific position.Tubercle can be as small as single cysteine residues.Tubercle can be a small peptide, as the residue around 8 residues of hCG β subunit 13.Tubercle also can be used as tubercle as β-Nei Xiananmei greatly to whole protein.Tubercle adds when protein synthesis, so just needn't remove any sealing residue or add any blocking group, is unlikely to cause protein denaturation.
Deformable β subunit tail end can be crosslinked with a generation of several α subunit residues, and wherein in these residues is substituted by a halfcystine; Find that based on this present invention has adopted a kind of new strategy probe or marker widely to be connected to the specific position of protein surface.Tubercle can be a fluorescin, as green fluorescent protein or other relevant molecules.They possess the feature of part or acceptor, can combine with other molecules.They can be proteolytic enzyme, toxin, antibody or antibody fragment, can make protein stride sequence, nucleic acid or the oligosaccharides of film translation as finding in the human immunodeficiency virus TAT protein.
In one embodiment, composition comprises protein portion, is positioned at the afterbody and the tubercle part of protein portion end; Wherein this protein portion contains alternative halfcystine in the need mark, and tubercle is positioned at a free end end of afterbody and contains cysteine residues, and the alternative cysteine residues of nodular cysteine residues and protein portion forms disulfide linkage.Term " protein portion " refers to any protein or polypeptide.The aminoacid sequence that term " afterbody " refers to one section sufficiently long, can make halfcystine in the tubercle and the alternative halfcystine in the protein portion form disulfide linkage.Afterbody can comprise proteinic part natural polypeptides in this protein portion, resembles among the hCG (see figure 1) the β subunit C-terminal, also can comprise the non-natural polypeptide that is connected to this protein portion end.Afterbody also should not contain the residue that can stop tubercle to be connected to alternative halfcystine, can be by the residue in the site of other protein bound as membrane spaning domain or generation.Term " tubercle " refers to the residue of halfcystine and this halfcystine either side, and wherein this cysteine residues is in the consecutive position of afterbody free-end.Tubercle can comprise one cysteine residues, one section linear aminoacid sequence that contains a halfcystine, one section merge with protein, linear aminoacid sequence that halfcystine is positioned at sequence, or the surperficial protein that contains cysteine residues.Tubercle can be designed for specific purpose or purposes, final mark or the probe that produces customization.For example, tubercle can be phenotypic markers thing, signal sequence, one section has sequence, enzyme or the target protein of high specific to being embedded in magnetic bead in the purifying chromatography column.
In another embodiment of the invention, the method at the specific site labelled protein is disclosed.This method comprise select to need the protein of mark, on desired protein the selection marquee site, and select required tubercle.Must contain a cysteine residues on the required tubercle.This method also comprises preparation coding desired protein, afterbody and required nodular construct.The desired protein of this construct coding comprises that in the need mark substitutes half a Guang amino-acid residue.With in this construct transfered cell, labelled protein is expressed then, wherein the halfcystine of halfcystine in the tubercle and desired protein forms disulfide linkage.
Term " construct " refers to include the nucleic acid carrier of the promotor that connects with the expressed sequence box, wherein this expressed sequence box a kind of particular proteins product of encoding.This construct has also comprised the sequence that is necessary that coded protein can be expressed, and any sequence of controllable express sequence box expression.These sequences are including, but not limited to promotor or initiating sequence, enhancer sequence, terminator sequence, RNA processing signal and/or polyadenylation signal sequence.Term " the expressed sequence box is expressed required sequence " refers to guarantee that the expressed sequence box produces the needed sequence of protein in rna transcription and translation subsequently.Term " promotor " refers to RNA polymerase combination, the needed section of DNA sequence of promotor gene rna transcription with it.Many well-known promotors are arranged in this area, and comprising can enhancing or controlling gene or expressed sequence box expression promoter.Construct of the present invention can be modified with PCR and cassette mutagenesis technology, to generate the nodular construct of coding desired protein.
The tubercle that is positioned at the protein specific position can be used for surveying the distance of being tried residue and acceptor or other protein contact surfaces.For example, can utilize tubercle to measure the degree of closeness of α subunit and receptor binding site among the hCG.Probe is connected to the residue that is positioned at binding pocket and will destroys in conjunction with active, points out this mark to be positioned at binding pocket.Using nodular another advantage of fixed point is that available bigger probe is differentiated near the residue protein-the be subjected to contact level.This exploration policy also can be used for phenotypic markers thing or signal sequence are connected to any required site of protein surface.In addition, protein probe also can be widely used in the fixing and protein target of protein.If the design of proteolytic enzyme recognition site is at deformable afterbody, this afterbody can cut away after crosslinked finishing, and makes probe catch (tethered) by the disulfide linkage of protein surface rather than by afterbody.
With the protein probe that method of the present invention produced, its purposes is not limited in the distance of inferring between protein loci.Targeting proteins enzyme if desired, only need existing fused protein preparation method (Sledziewski etc., 6,300, No. 099 United States Patent (USP)) changes a little, simply the encoding sequence of proteolytic enzyme and 5 ' or 3 ' end of this protein coding sequence are merged, just many different proteolytic enzyme can be connected to this proteinic N-terminal or C-terminal.Unfortunately, proteolytic enzyme can make this protein be destroyed near protein.
Use the protein tubercle to solve this problem, because adopt proteinic strategy that tubercle is connected to described herein, proteolytic enzyme can be fixed on the position that can not attack the molecule that is connected.And, proteolytic enzyme towards cracking that can the required substrate of catalysis, as the cracking of acceptor.Available method described herein is designed into proteolytic enzyme on the hCG, so that this proteolytic enzyme has precedence over other any protein with the cracking of LH acceptor, makes it lose the metakentrin activity.This protease protein matter tubercle can serve as the ideal methods of treatment in the treatment of polycystic ovary syndrome, and nearly has 1/3rd to be caused by polycystic ovary syndrome in all human infertility.
In another embodiment of the invention, the compositions and methods of the invention can be used for promoting two proteinic stable associations.Here the data presentation that provides crosslinked hCG protein analogue when low pH is much more stable than natural hCG.The introducing of disulfide linkage makes stability rising [Matzuk, the M.M.﹠amp of heterodimer between the hCG subunit; Boime, I. (1988) J.Cell Biol.106,1049-1059; Heikoop, J.C.; Van, den boogaart; Mulders, J.W.; Grootenhuis, P.D., (1997) are based on the protein engineering of disulfide linkage between subunit in structure Design and the luteinizing hormone-releasing hormone (LRH), Nature Biotechnology 15:658-662].In the past, once be that disulfide linkage between subunit is introduced on the basis in protein with the crystalline structure.The invention discloses when high resolution structures can't obtain during the protein synthesis with subunit between disulfide linkage introduce method between two proteins.
In another embodiment of the invention, composition and method can be used for promoting the stable association of archaeal dna polymerase and DNA.What can estimate is, introduces to be looped around around the DNA and can to make polymerase stabilization to DNA with the connector that disulfide linkage is fixed in polysaccharase, and the length of last transcription product increases thereby make eventually.
In another embodiment of the invention, mixture of the present invention and method can be used for generating the protein heterodimer that is made of the sugar-protein hormone that lacks one or more oligosaccharides.People wish that the disappearance of glycosylation signal in second ring of hCG α subunit can reduce the secretion heterogenous dimeric ability of Mammals, and stimulating organism is replied (Einstein, M., Lin, W., Macdonald, G.J.﹠amp; Moyle, W.R. (2001) Exp.Biol.Med.226,581-590; Slaughter, S., Wang, Y.H., Myers, R.V.﹠amp; Moyle, W.R. (1995) Mol.Cell.Endocrinol.112,21-25; Yen, S.S.C., Llerena, O., Little, B.﹠amp; Pearson, O.H. (1968) J.Clin.Endocrinol.Metab.28,1763-1767; Matzuk, M.M.; Boime, I., (1989), mutagenesis and transgenosis are in the effect of defining luteinizing hormone-releasing hormone (LRH) oligosaccharides specific site, Biol.Reprod.40:48-53).As shown here, the coexpression of hCG β S138C and α N52C makes the output of heterodimer can be suitable with hCG, wherein α N52C be in the α subunit Asn52 residue by halfcystine alternate α subunit analogs.The sudden change of this α subunit makes the glycosylation blackout, and the hCG analogue that causes oligosaccharides in second ring of efficiency ratio α subunit of hCG analogue to be removed by glycanase digestion is much higher.
In another embodiment, disclose and utilized the compositions and methods of the invention to promote the method that the protein polymer forms, wherein avidity is minimum or do not have avidity to each other for the subunit in this polymer.For example, the data declaration that provides among Fig. 6 might be connected to β-Nei Xiananmei one in several different loci among the hCG, and known β-Nei Xiananmei is not relevant with hCG.By introducing protease cracking site (cutting β subunit C-terminal) on the β of hCG subunit and the securing band between the halfcystine, but the heterodimer that makes preparation β-Nei Xiananmei or other protein almost stables be connected in any site of hCG becomes possibility.
In another embodiment, the halfcystine on the tubercle can move on to the surface location of tubercle part.So just allow tubercle on ideal direction and site, directly to be connected with protein.
In another embodiment, available method of the present invention is carried out mark with protein with the phenotypic markers thing.The phenotypic markers thing often is connected on the protein, so that the detection of protein-protein or protein-macromolecule interaction.In the past, the phenotypic markers thing is connected to proteinic N-terminal or C-terminal.Yet many phenotypic markers things are only effective at a proteinic end.And when protein terminal participated in this proteinic function, the practicality that is added to the phenotypic markers thing of protein terminal descended greatly.Method of the present invention can make the phenotypic markers thing be positioned the outer site of isolating protein end, makes the phenotypic markers thing more useful.Introducing cracking site at afterbody can make proteinic free end and not destroy this phenotypic markers thing.
In another embodiment of the invention, can introduce aldehyde radical at proteinic privileged site.Aldehyde radical be do not exist usually in the protein, unusual a kind of reactive group of ideal, and can be used for the reactant that several are different, be connected to proteinic surface as fluorophor.Present method has been utilized N-terminal Serine or the threonine residues well-known reactivity that periodate oxidation had (Yoo, J., Ji, the I.﹠amp to gentleness; Ji, T.H. (1991), J.Biol.Chem.266,17741-17743; Geoghegan, K.F., Stroh, J.G., (1992), and fixed point takes place by 2-amido alcoholic acid periodate oxidation with peptide and protein and combines in non-peptide group, the application that Serine N-is end modified, Bioconjug.Chem.3:138-146).Like this, can behind enzymatic lysis, immediately serine residue be introduced proteinic cracking site.For example, can be incorporated into aminoterminal Serine by the site that enteropeptidase is discerned with one immediately, make the aminoterminal of halfcystine that tubercle is crosslinked in need marker site part and target halfcystine.To produce sequence X like this
1-Asp-Asp-Asp-Asp-Lys-Ser-Y
m-Cys-Z
n(SEQ ID NO:56), X, Y and Z refer to the arbitrary amino acid of afterbody, l, m and n refer to the amino acid whose length of this afterbody.This proteinic generation causes the halfcystine of afterbody to be linked to required site on the protein, and wherein this site has replaced to halfcystine.Enteropeptidase cracked result produces the terminal Serine that is easy to be handled by the periodate of gentleness institute's oxidation.The aldehyde radical that is generated is easy to react with various hydrazides derivative compounds, comprises various fluorophors and vitamin H.This method very is particularly useful when protein does not contain unconjugated halfcystine.
In another embodiment, the method for utilizing composition of the present invention to come specific site on the closed protein matter is disclosed.For example, with toxin or enzyme may cell killing reactive site to seal before its activity of needs be unusual ideal.Cancer therapy may be to use an extraordinary field of the present composition and method.Thereby, can introduce a halfcystine near the position enzyme or the toxin reactive site.The tubercle that will contain halfcystine be connected to can be near reactive site halfcystine form the protein amino end or the C-terminal of disulfide linkage, can stop the interaction of reactive site and its target.Tubercle can comprise the target protein that merges with afterbody, and wherein this afterbody makes enzyme/toxin-target protein mixture and specific target anchor schedule cell surface.Protease treatment mixture with the cracking afterbody will make the reactive site of enzyme or toxin come out.This strategy can be used for shielding the activity of enzyme or toxin, contains the position of the enzyme of cleavable afterbody up to its arrival, thereby toxin exposure is come out.For example, such situation can take place after in the cell in the mixture internalization.
Protein is mutually compound to each other under the normal circumstances, and nodular another purposes is this unnecessary association that stops between protein.Like this, disulfide linkage can be designed to tubercle is fixed in two positions between the protein contact surface.
In another embodiment, the invention provides a kind of method of utilizing the minority step that the target protein clean cut separation is come out.Adopt method as described herein to make up coding proteins of interest matter, afterbody and nodular expression construct.The position of target halfcystine can be in the lip-deep any required site of C-terminal, N-terminal or proteins of interest matter on the proteins of interest matter.After construct is expressed,, the protein-tubercle mixture that is produced is flow through chromatography column rapidly in case the coupling halfcystine on the tubercle combines with halfcystine on the desired protein.By selecting the suitable tubercle of combining closely, protein-tubercle and affine resin-bonded, unconjugated protein and cellular component are by wash-out.Then,, cut tubercle, only keep the protein of purifying the mixture wash-out.For example, can use (Strategene ' s Affinity
TM) the pCAL carrier, select for use calmodulin matter binding peptide (CBP) as tubercle simultaneously.CBP-tubercle and affine resin-bonded, the EDTA of available 2mM is wash-out under neutral pH, avoids using the harsh elution requirement that may make protein denaturation.But the present invention can utilize many possible carriers and tubercle combination.
The present invention relates to make up protein of interest matter so that the protein tubercle possesses a short tail that contains cleavage site aspect another of protein purification.The tubercle tail must be enough short, makes this tubercle not form disulfide linkage with protein itself and link.On the contrary, the structure of this protein and afterbody should make tubercle and another protein form disulfide linkage.In solution, there is the protein of afterbody to resemble and presents linear array a string pearl, be connected with the formed disulfide linkage of halfcystine on second proteinic protein portion by the halfcystine on the first proteinic tubercle between pearl.Halfcystine on halfcystine on second proteinic tubercle and the 3rd the proteinic protein portion forms disulfide linkage, or the like.The end last protein chain that is produced can carry out sucrose gradient centrifugation, and protein chain will be settled down to the bottom of gradient because of its weight.At last, for protein chain and other settled materials, separate as the cracked cytolemma, protein chain place bottom is handled with the enzyme of specificity cracking short-tail portion, produces one protein.Mixture is centrifugal again.One protein will be parked in the position of gradient near the top, be easy to collect and purifying.Well-known method of purifying protein comes purifying in the also available aforementioned additive method of protein chain or this area.
Another aspect of the present invention is to make up protein of interest matter, so that each protein tubercle and another protein tubercle form latticelike structure.Similar with the afterbody in the pearl string method, the structure of afterbody should make tubercle not react with the halfcystine on the protein portion.Protein also can all comprise afterbody and tubercle at proteinic two ends.Protein portion should comprise a plurality of half Guang amino-acid residues that substitute, so that can form disulfide linkage with protein from single proteinic a plurality of tubercles.The quantity and the position that substitute halfcystine on the protein tubercle should be determined with computer simulator or additive method in advance.In solution,, thereby be the grid spline structure because one protein can form disulfide linkage with a plurality of protein.Protein tubercle matrix can be with above-mentioned any method purifying, and please sincerely remember: the molecular weight of structure is big more, and then it more may be as the good candidate thing of centrifugation technique.
In another embodiment, the present invention is used in and adds halfcystine on the protein.Usually utilize halfcystine to be connected to protein surface and other molecules such as fluorophore be connected to contain the proteinic unique reactive of second half Gelucystine, halfcystine is introduced in the protein.Numerous protein contains halfcystine or disulfide linkage, make to utilize introduces halfcystine in the molecule difficulty that becomes.Tryptophane is introduced into the afterbody that is close to halfcystine, thereby walks around this difficulty, wherein this halfcystine is used for tubercle and the protein that needs to modify crosslinked in introducing halfcystine part.Because the absorption spectrum of tryptophane, tyrosine and phenylalanine is different, the rayed of 295nm wavelength is the tryptophan residue in the targeting proteins matter optionally.To cause the destruction of contiguous disulphide like this, and make the sulfydryl on the halfcystine have reactive behavior.Adding under the condition that proteinic group exists, protein is shone and will mark in the required part of protein, even protein contain several other disulphide also can mark on.Even now is beneficial near the tryptophan residue other disulphide and also becomes and have reactive behavior, but in fact most protein contains the only a few tryptophane, and this fact means that this is not problem usually.
In another embodiment, the present invention can be used for producing and obtaining the heterodimer protein of high yield.When making up the nodular expression construct of protein, a dimer in the heterodimer can comprise this protein portion.If this dimeric protein portion does not have naturally occurring afterbody, afterbody can be merged in the dimeric protein portion.Tubercle can comprise other dimers of heterodimer.When construct was expressed, owing to connect the existence of dimer afterbody and the ability of formation disulfide linkage, the possibility that forms heterodimer was bigger.The dimer that this method has been avoided heterodimer forms the problem of homodimer during coexpression in same cell.
Existing (Bemard, M.P., Myers, the R.V.﹠amp of describing in the source of used hCG and antibody in these researchs; Moyle, W.R. (1998) Biochem.J.335,611-617; Moyle, W.R, Campbell, R.K., Rao, S.N.V., Ayad, N.G., Bernard, M.P., Han, Y.﹠amp; Wang, Y. (1995) J.Biol.Chem.270,20020-20031; Moyle, W.R., Matzuk, M.M., Campbell, R.K., Cogliani, E., Dean Emig, D.M., Krichevsky, A., Barnett, R.W.﹠amp; Boime, I. (1990) J.Biol.Chem.265,8511-8518.).Carry out cassette mutagenesis between the natural A paI site of hCG cDNA and BamHI site, preparation can be expressed the construct of hCG β-S138C, wherein the BamHI site as design as described in the document at the downstream of terminator (Campbell, R.K., Dean Emig, D.M.﹠amp; Moyle, W.R. (1991) Proc.Natt.Acad.Sci. (USA) 88,760-764; Campbell, R.K., Dean Emig, D.M.﹠amp; Moyle, W.R. (1991) Proc.Natt.Acad.Sci. (USA) 88,760-764.).Be used to express carrier (Xing, Y., Lin, W., Jiang, M., Myers, R.V., Cao, D., Bernard, the M.P.﹠amp that substitutes halfcystine α subunit by the described method preparation of document simultaneously; Moyle, W.R., the prolan analogue that other modes are folding: folding and bioactive prompting to hormone.Journal?of?Biological?chemistry.2001.)。Coding people's α subunit or halfcystine substitute the construct of analogue and press described method of document and hCG β subunit or hCG β-S138C coexpression (Campbell, R.K., Dean Emig, D.M.﹠amp in the COS-7 cell; Moyle, W.R. (1991) Proc.Natt.Acad.Sci. (USA) 88,760-764.).α subunit antibody A113 that employing is used to catch and the radiation iodate β subunit antibody B110 that is used to detect use the sandwich immunoassay to measure material (Moyle, W.R., Ehrlich, the P.H.﹠amp that is secreted in the substratum; Canfield, R.E. (1982) Proc.Natt.Acad.Sci. (USA) 79,2245-2249.).Also it is handled the heterodimer that promote to lack disulfide bond crosslinking dissociate (Xing, Y., Lin, W., Jiang, M., Myers, R.V., Cao, D., Bernard, M.P.﹠amp with acid pH with the described method of document; Moyle, the prolan analogue that other modes of W.R. are folding: folding and bioactive prompting to hormone.Journalof?Biological?chemistry.2001.)。Such by what reported in the past, right with Chinese hamster cell (CHO) the monitoring analogue of overexpression rat LH R
125I-hCG and LHR binding ability and stimulation cyclisation AMP gather influence (Bernard, M.P., Myers, the R.V.﹠amp of ability; Moyle, W.R. (1998) Biochem.J.335,611-617; Moyle, W.R., Matzuk, M.M., Campbell, R.K., Cogliani, E., Dean Emig, D.M., Krichevsky, A., Barnett, R.W.﹠amp; Boime, I. (1990) J.Biol.Chem.265,8511-8518; Moyle, W.R., Campbell, R. K., Myers, R.V., Bernard, M.P., Han, Y.﹠amp; Wang, X. (1994) Nature 368,251-255.).
Explain that interactional another alternative route of hCG-LHR comprises surface (Moyle, W.R., Matzuk, M.M., the Campbell that differentiates that this hormone exposes in hormone-receptor complex, R.K., Cogliani, E., Dean Emig, D.M., Krichevsky, A., Barnett, R.W.﹠amp; Boime, I. (1990) J.Biol.Chem.265,8511-8518.).By elimination methods, the surface of area exposed in hormone-receptor complex being surveyed and drawn this hormone is to disclose the position that can contact with acceptor.Since these data are collected in this hormone still keeps itself and the active research of acceptor interaction, thereby interact and change the easier explanation of data that is obtained than depending on hormone receptor.Most of method that detects the noncontact residue depends on the use of monoclonal antibody probe, and this method is subjected to the strict restriction of resolving power.For walking around this restriction, can measure activity (Xing, Y., Lin, W., Jiang, M., Myers, R.V., Cao, D., Bernard, the M.P.﹠amp of the analogue that is pinned by securing band; Moyle, the prolan analogue that other modes of W.R. are folding: folding and bioactive prompting to hormone.Journal?of?Biologicalchemistry.2001.)。May block the hormone receptor interaction although contain the zone of part securing band and C-terminal connection, several analogues in these analogues have the basic activity identical with hCG.Securing band linked with the α subunit may change dimeric conformation, this phenomenon may can explain why active low some analogues are.Recently, the long C-terminal that the someone finds to upset in the β subunit can sufficient movement, can scan the most surfaces of heterodimer, up to introduce at the 138th residue halfcystine and with introduce halfcystine in the α subunit and form disulfide linkage till.Securing band in these analogues tacks as the securing band among the hCG, makes this sudden change be difficult to influence the conformation of heterodimer.As described here, the analogue that has those securing band of many specific activitys to be connected on the β subunit in these analogues is eager to excel.The result of the research of implementing shows that also the major part of second ring of hCG α subunit does not contact with LHR, and second ring may be near being subjected to contact level.We have compared the hCG analogue before halfcystine that the β of confusion subunit C-terminal is introduced by second ring and α subunit C-terminal tack and activity (seeing Fig. 2 and Fig. 3) afterwards.In LHR combination and signal conduction analysis, except halfcystine was positioned at the most advanced and sophisticated analogue of second ring, all the other lacked crosslinked heterodimer activity and are at least 25% of hCG.This results suggest, little residue is made part contribution to hCG-LHR in conjunction with total energy in arbitrary zone in two zones, and this point thinks that with those C-terminal is that the necessary report of usefulness conflicts mutually.β subunit C-terminal probe is fastened to second ring of α subunit in the face of first ring of β subunit and the 3rd ring, and relative less with the influence of signal conduction to combination, this part of prompting hormone unlikely contacts with acceptor.β subunit C-terminal and other residue are linked and cause loss of activity, and these parts of prompting α subunit may be approaching with acceptor.Interaction with this method research hFSH and fsh receptor shows that hFSH passes through similar generally mode and combines with fsh receptor, but facilitates the hFSH part of key receptor contact different.Can these observationss have also been proved conclusively with LH acceptor and the chimeric hCG-hFSH part of fsh receptor bonded simultaneously with this method research.Therefore, compare with the degree that FSHR is contiguous, different piece and LHR in the mosaic are more contiguous.Differentiate when other protein do not participate in the part of protein-protein contact, adopt the similar mutagenesis strategy should be very useful.
Research described herein has also been demonstrated with this method and has been differentiated hFSH and fsh receptor bonded binding site, and the interaction of difunctional hCG/hFSH analogue and LH acceptor and fsh receptor is contrasted.HFSH does not have " securing band " (afterbody) (Fig. 8, SEQ ID NO:41), therefore prepared the hFSH subunit analogs that hCG β subunit C-terminal (fqdsssskapppslpspsrlpgpstdpilpg, SEQ ID NO:55) part residue is arranged in β subunit C-terminal place coding.With implement to substitute the 138th serine residue with halfcystine in the research with hCG similar, substitute the 132nd serine residue (Fig. 8, SEQ ID NO:42) of hFSH β subunit analogs with halfcystine.Then hFSH β subunit S132C is expressed with the several α subunit analogs that contain an alternative halfcystine.As estimating according to the experience of hCG analogue, this heterodimer is much more stable than natural hFSH when hanging down pH, proves that the β subunit of hFSH analogue and α subunit take place crosslinked by disulfide linkage.Many very high activity (Figure 13) that have in the hFSH receptor assay are arranged in these analogues.Compare with hCG deutero-analogue, the hFSH derived analogs shows some differences in determination of activity.These differences show, the interaction of HSH and fsh receptor is different with the interaction of hCG and LH acceptor, thereby also difference of the conformation model of each hormone-acceptor.The result shows that also hCG β subunit C-terminal sequence can serve as " afterbody ", and tubercle is added on the protein surface that lacks suitable site or " afterbody "." afterbody " sequence needs sufficiently long so that the halfcystine in the afterbody can get at the halfcystine that protein surface will connect, and this sequence can not contain and stop the residue of this sequence near the protein surface halfcystine simultaneously.Thereby thereby stop this sequence near the residue of protein surface halfcystine comprise those residues that sequence are folded into can make the isolation structure territory of this halfcystine shielding, such as contain protein is become be connected in cytolemma make the signal of this halfcystine shielding residue, contain the residue that makes protein and another protein bound make the signal that this halfcystine shields, or altitudinal belt electric charge and interactional residue between the blocking protein surface.
The part of sugar-protein hormone securing band can be exchanged mutually, prepare the difunctional analogue (Moyle etc., NO.5,508, No. 261 United States Patent (USP)s) of sugar-protein hormone with this.Interact in order further to distinguish metakentrin, as the difference of hCG and LH acceptor interaction; Interact with follicle stimulating hormone, as hFSH and the interactional difference of fsh receptor, we prepared known can while and LH and a kind of hCG analogue of fsh receptor bonded.The 101-114 position residue of hCG is that hFSH 95-108 position residue (Fig. 8, SEQ ID:38) substitutes with the corresponding part of its hFSH.As the way of front hCG and hFSH analogue, the 38th serine residue of this analogue C-terminal substitutes (Fig. 8, SEQ ID:39) with halfcystine.Construct is contained the analogue that substitutes halfcystine with several expresses in the COS-7 cell.Use the sandwich immunoassay, adopt the α subunit antibody of hCG and β subunit antibody that the heterodimer that produces is carried out quantitatively.Stability raises during based on low pH, finds that many heterodimers take place crosslinked by disulfide linkage between α subunit and β subunit.Compare with the fsh receptor interphase interaction with LH, the interaction of a part has tangible difference in these analogues.For example, crosslinked analogue takes place in the 37th cysteine residues of β subunit C-terminal and α subunit, can interact well with the LH acceptor, but can not be fully and fsh receptor interact (Figure 12-14).The interaction that this point has been proved conclusively hCG and LH acceptor obviously is different from the interaction of hFSH and fsh receptor, and further provides important support for the model of each hormone and its acceptor interaction.
The result
HCG α subunit analogs and natural hCG β subunit or hCG β-S138C
The COS-7 cell of carrier cotransfection of the carrier of most of α subunit analogs and coding natural β subunit (table 1) or hCG β-S138C (table 2) of encoding can assemble the allos aggressiveness and it is secreted in the substratum.Secretion seldom or not excretory heterodimer comprises that those substitute the analogue (table 1) of Tyr37, Pro40, Asn52 and Y89C residue in β subunit and the α subunit with halfcystine.α N52C/ β secretion may reflect that seldom it lacks the glycosylation signal of N link, and this signal is that heterodimer is effectively secreted institute essential (Matzuk, M.M.﹠amp under the normal circumstances; B, I. (1988), J.Cell Biol.106,1049-1059).
Table 1, usefulness are specified the heterodimer output of the COS-7 cell of subunit construct transfection
| Transfection | Dimer content in the substratum (ng/50 μ l) mean ± SEM | ||
| ???αG22C+β | ????9.034 | ??± | ?0.414 |
| ???αY37C+β | ????0.464 | ??± | ?0.052 |
| ???αP38C+β | ????1.710 | ??± | ?0.026 |
| ???αP40C+β | ????0.588 | ??± | ?0.093 |
| ???αL41C+β | ????7.617 | ??± | ?0.438 |
| ???αR42C+β | ????0.962 | ??± | ?0.135 |
| ???αS43C+β | ????3.571 | ??± | ?0.156 |
| ???αK44C+β | ????2.788 | ??± | ?0.358 |
| ???αK45C+β | ????1.090 | ??± | ?0.093 |
| ???αT46C+β | ????7.044 | ??± | ?0.229 |
| ???αM47C+β | ????9.491 | ??± | ?0.524 |
| ???αL48C+β | ????1.497 | ??± | ?0.170 |
| ???αV49C+β | ????2.579 | ??± | ?0.297 |
| ???αQ50C+β | ????1.985 | ??± | ?0.153 |
| ???αN52C+β | ????0.615 | ??± | ?0.081 |
| ???αV53C+β | ????6.108 | ??± | ?0.356 |
| ???αM71C+β | ????6.153 | ??± | ?0.332 |
| ???αG73C+β | ????2.849 | ??± | ?0.194 |
| ???αT86C+β | ????4.973 | ??± | ?0.027 |
| ???αY88C+β | ????7.930 | ??± | ?0.290 |
| ???αY89C+β | Do not detect | ||
| ???αH90C+β | ????0.971 | ??± | ?0.170 |
| ???αK91C+β | ????1.049 | ??± | ?0.055 |
| ???αS92C+β | ????2.595 | ??± | ?0.106 |
Contain in the heterodimer (table 2) of hCG β-S138C and can detect most α subunit analogs.The few analogue of secretion comprised Tyr37, Pro40 and Asn52 when hCG β-S138C can promote to form some and natural β subunit co expression, but do not comprise Tyr89 (table 2, Fig. 1).These analogues have many analogues to have crosslinked between subunit in the table 2, and this is because can detect easily with low pH simple process.The heterodimer that contains natural α subunit or α G22C and α V53C α subunit analogs is destroyed when hanging down pH, points out the disulfide linkage between their shortage subunits.But the heterodimer that contains α Q5C, α Q27C, α P40C, α K51C, α L41C, α M71C and α V76C α subunit analogs has only sub-fraction to demonstrate by disulfide linkage between subunit stable (table 2).For the analogue that seldom or not forms heterodimer, the halfcystine in its α subunit analogs is positioned at subunit contact surface or far apart with the Asp111 residue of β subunit, and this residue is first residue that β subunit C-terminal extends.Therefore, as if the 138th cysteine residues of β subunit is near being obstructed during halfcystine on the α subunit.This phenomenon points out the disulfide bond crosslinking between most of subunit to form when subunit is assembled into structure and the similar heterodimer of hCG.Tried only to have in the α subunit analogs analogue neither also not form heterodimer with hCG β-S138C with hCG β subunit, it contains an alternate halfcystine at the Tyr89 place.Although other residues except that halfcystine can be removed or be replaced into to this tyrosine, and do not destroy structure (Pierce, the J.G.﹠amp of heterodimer; M.50, Parsons, T.F. (1981) Annu.Rev.Bioche 465-495), are not that the folding institute of α subunit is essential, substitute pleated sheet structure (Chem, F., Wang, the Y.﹠amp that this tyrosine still may destroy the α subunit with halfcystine; Puett, D. (1992) Mol.Endocrinol.6,914-919).
Table 2, used appointment α subunit construct and hCG β-S138C
| The α subunit analogs | Heterodimer (ng/50 μ l) | Crosslinked (%) a | Securing band is fixed (B11/B110) b |
| Natural α | ????26.2±0.8 | ????-6.5±2.6 | Do not calculate |
| ????αQ5C | ????14.3±1.3 | ????17.5±9.9 | ??????0.98 |
| ????αG22C | ????9.1±0.4 | ????-3.1±1.0 | Do not calculate |
| ????αQ27C | ????3.3±0.0 | ????21.1±4.2 | ??????1.03 |
| ????αR35C | ????6.0±0.0 | ????98.4±4.9 | ??????0.97 |
| ????αY37C | ????5.4±1.2 | ????80.2±0.5 | ??????0.18 |
| ????αP38C | ????0.3±0.04 | Not check | Do not calculate |
| ????αP40C | ????2.1±0.04 | ????52.8±4.3 | ??????0.64 |
| ????αL41C | ????3.2±0.04 | ????37.2±0.6 | ??????0.49 |
| ????αR42C | ????22.1±5.3 | ????67.7±5.3 | ??????0.48 |
| ????αS43C | ????29.7±1.4 | ????81.3±1.9 | ????0.60 |
| ????αK44C | ????4.0±0.1 | ????55.4±1.3 | ????0.84 |
| ????αK45C | ????5.8±0.1 | ????82.9±6.2 | ????0.85 |
| ????αT46C | ????21.4±0.4 | ????76.8±0.6 | ????1.14 |
| ????αM47C | ????4.9±0.8 | ????72.0±2.0 | ????0.82 |
| ????αL48C | ????14.3±0.9 | ????88.5±2.1 | ????0.86 |
| ????αV49C | ????6.0±0.5 | ????70.5±6.1 | ????0.76 |
| ????αQ50C | ????0.8±0.1 | ????66.7±7.4 | ????0.82 |
| ????αK51C | ????4.1±1.2 | ????27.5±1.4 | ????0.86 |
| ????αN52C | ????6.3±0.2 | ????70.6±3.9 | ????0.88 |
| ????αV53C | ????6.1±0.4 | ????4.3±2.9 | ????1.01 |
| ????αS64C | ????4.4±0.3 | ????82.5±8.9 | ????0.85 |
| ????αM71C | ????6.2±0.3 | ????14.1±3.2 | Do not calculate |
| ????αV76C | ????4.3±0.0 | ????41.9±4.9 | ????1.10 |
| ????αT86C | ????7.6±0.8 | ????88.4±9.3 | ????0.66 |
| ????αY88C | ????7.4±0.3 | ????83.0±6.2 | Do not calculate |
| ????αY89C | Can not detect | Do not check | Do not calculate |
| ????αH90C | ????7.2±1.3 | ????79.2±1.0 | ????0.86 |
| ????αK91C | ????9.9±5.7 | ????69.4±3.3 | ????0.91 |
| ????αS92C | ????15.1±0.8 | ????140.7±14.4 | ????0.98 |
A) this value is handled the material percentage ratio calculating that still retains in the sample of back with the described acid pH of body part.
B) this value is to handle the back with low pH to reach with A133
125I-B111 in sandwich is analyzed measured activity with A113 and
125The measured active ratio of I-B110.Detect any B111 combination, the prompting securing band is fixed.Observed value is on the low side in some cases may reflect the influence of β subunit C-terminal to B111 and the interactional ability of crosslinked heterodimer.
Many halfcystines in second ring of α subunit residue substitute to the receptors bind of hCG and signaling activity influence very little (table 3, Fig. 2).With halfcystine substitute α Met47 position (table 3, Fig. 2) and α Lys51 position (Einstein, M., Lin, W., Macdonald, G.J.﹠amp; Moyle, W.R (2001) Exp.Biol.Med.226,581-590.) α subunit residue makes the active decline (table 3) relatively of the specific activity hCG of heterodimer in combination and signal conduction analysis.And α Lys51 is by active descend greatly (Einstein, M., Lin, W., Macdonald, the G.J.﹠amp of comparing with hCG of L-Ala alternate analogue; Moyle, W.R. (2001) Exp.Biol.Med.226,581-590.), α Met47 by L-Ala alternate analogue hCG-α M47A activity in two analyses all almost with hCG quite (table 3).The existence of the 47th methionine(Met) is not to keep the hCG activity necessary in the prompting α subunit residue.The specific function of α Lys51 in acceptor interaction still has to be determined.The 99th residue of the 51st residue of α subunit and β subunit had stronger activity than those α Lys51 by halfcystine or L-Ala alternate heterodimer with the heterodimer of disulfide bond crosslinking, find based on this, substitute the conformation that α Lys51 side chain probably changes heterodimer.
Table 3, second ring of α subunit and C-terminal sudden change are to the active influence of heterodimer metakentrin
| The α subunit analogs | The usefulness of natural β subunit analogs (hCG percentage ratio, 95%CL) | Acid stablize hCG β-S138C analogue usefulness (hCG percentage ratio, 95%CL) | ||
| Receptors bind a | The signal conduction a | Receptors bind a | The signal conduction a | |
| ???αR42C | ??113(97-132) | ??103(85-124) | ????327(305- ????351) | ????332(287- ????385) |
| ???αS43C | ???35(31-39) | ??74(50-110) | ????17(15-20) | ????43(37-49) |
| ???αK44C | ???66(60-72) | ??97(75-124) | ????250(228- ????274) | ????191(148- ????247) |
| ???αK44A | ???84(71-101) | Untransfected | Untransfected | |
| ???αK44E， ????K45Q | ???58(54-61) | ??42(32-55) | Untransfected | Untransfected |
| ???αK45C | ???93(87-100) | ??91(71-117) | ????223(212- ????257) | ????168(129- ????217) |
| ???αT46C | ???38(35-42) | ??56(43-73) | ????50(44-57) | ????52(43-64) |
| ???αM47C | ???8.7(7.4-10) | ??23(16-32) | ????4.1(2.9-5.9) | ????2.4(2.1-2.7) |
| ???αM47A | ???83(71-97) | Untransfected | Untransfected | |
| ???αL48C | ???60(54-68) | ??147(96-226) | ????66(60-72) | ????99(81-120) |
| ???αV49C | ???70(64-77) | ????133(102- ????174) | ?????71(65-76) | ????99(84-119) |
| ???αQ50C | ???81(76-85) | ????216(163- ????285) | ????308(241- ????395) | |
| ???αK51C | Detection less than * | Detection less than * | ||
| ???αN52C | Do not do | Do not do | ?????61(57-65) | |
| ???αV53C | ???149(137- ???162) | Do not do | Do not do | |
| ???αS64C | Do not do | Do not do | ?????89(78-101) | |
| ???αT86C | ???95(84-106) | ????444(354- ????555) | (2.2 intermediate) | ????0.18(0.16- ????0.21) |
| ???αY88C | ???141(128- ???156) | ????74(64-84) | (0.05 intermediate) | ????0.12(0.09- ????0.16) |
| ???αY89C | Do not do | Do not do | Do not do | Do not do |
| ???αH90C | ???30(26-34) | ????65(48-89) | ?????3.1(1.8-5.4) | ????1.0(0.96- ????1.1) |
| ???αK91C | ???41(38-45) | ????56(46-68) | ?????2.2(1.1-4.4) | Can not detect |
| ???αK91E | ???4.3(3.4-5.5) | ????1.1(0.1-2.2) | Untransfected | Untransfected |
| ???αK91M | ???68(36-144) | ????66(45-140) | Untransfected | Untransfected |
| ???αS92C | ???100(91-110) | ????38(31-47) | ?????28(23-34) | ????13(12-14) |
A) the analogue concentration of measuring based on the sandwich immunoassay.The IC50 value that these values are summed up by test is calculated and is got.Because the amount that the transfection COS-7 of institute cell generates very little, has several analogues not do check.As shown in table 1, α/hCG β-S138C does not obtain any stable heterodimer after acid treatment.
There is report to claim: to make the α Lys44 in second ring of α subunit into L-Ala and make hCG active descend 100 times or more (Xia, H., Chen, F.﹠amp; Puett, D. (1994) Endocrinol.134,1768-1770).And much smaller (table 3) that our discovery is estimated the active influence ratio of hCG with α Lys44 in the alternative α subunit of halfcystine and near several residues thereof.(Slaughter, S., Wang, Y.H., Myers, R.V.﹠amp in the irrelevant research process of part that check is predicted the subunit associating back vertical electric charge of α subunit second ring; Moyle, W.R. (1995) Mol.Cell.Endocrinol.112 21-25), prepares the 44th and the 45th Methionin and uses glutaminate and glutamine alternate analogue (hCG-α K44E, K45Q) (Figure 15, SEQ ID NO:52) respectively.Unexpectedly, this analogue all has in two mensuration and hCG-α K44A (Figure 15, SEQ ID NO:51) and the identical activity of hCG-α K44R (Figure 15, SEQ ID NO:53), wherein both α Lys44 of back substituted by L-Ala and arginine (table 3, Figure 19).The high reactivity of back one analogue can draw (Piece, J.G.﹠amp by the fact prediction that contains identical alternate horse α subunit; Parsons, T.F. (1981) Annu.Rev.Biochem.50,465-495).These researchs are with consistent with alternative these residue gained result of halfcystine, contradict (Xia, H., Chen, F.﹠amp with in early time discovery; Puett, D. (1994) Endocrinol.134,1768-1770), in prompting α subunit second ring on the minor spiral all non-LHR of positively charged tyrosine residues of the high conservative institute that interacts essential.
The removal of β Cys26
Found if the C-terminal of securing band is attached to the β Cys110 of β subunit core, made itself and the 26th residue of β subunit form disulfide linkage and be obstructed that then this residue can form crosslinked (Xing with the halfcystines in many introducing α subunit analogs, Y., Lin, W., Jiang, M., Myers, R.V., Cao, D., Bernard, M.P.﹠amp; Moyle, the prolan analogue that other modes of W.R. are folding: folding and bioactive prompting to hormone.Journal?of?Biologicalchemistry.50，46953-46960，2001.)。This discovery changes the position of securing band, and the phenotype of antibody B111 is disappeared.For whether the securing band of learning crosslinked analogue links to each other with β Cys26, be that the ability that antibody B111 and antibody B110 are discerned is contrasted to these analogues, this antibody can be discerned different β subunit phenotypes.As shown in table 2, each crosslinked heterodimer all can be discerned by B111, although not necessarily discerned by B110 equally, the prompting securing band links to each other with β Cys26 with pattern identical among the hCG.Although we can not get rid of the possibility that β Cys110 links to each other with the halfcystine of introducing in some analogue α subunits, this possibility almost is non-existent.Reason has 2 points.The first, used β subunit all contains a halfcystine at the 26th residue in these researchs.The disappearance of the 26th residue halfcystine is essential for making in securing band and the α subunit halfcystine of introducing form crosslinked, no matter its position how (Xing, Y., Lin, W., Jiang, M., Myers, R.V., Cao, D., Bernard, M.P.﹠amp; Moyle, the prolan analogue that other modes of W.R. are folding: folding and bioactive prompting to hormone.Journal?of?Biological?chemistry.2001.)。The second, the halfcystine position that the α subunit least may be discerned by B111 in the crosslinked analogue is near the binding site (Fig. 1) of B111.The crosslinked position stability of these analogues β subunit C-terminal that makes of these promptings is in the position of mediation B111 contact heterodimer.
α subunit C-terminal
α subunit C-terminal is considered to LHR interaction institute essential (Piece, a J.G.﹠amp; Parsons, T.F. (1981) Annu.Rev.Biochem.50,465-495; Yen, S.S.C., Llerena, O., Little, B.﹠amp; Pearson, O.H. (1968) J.Clin.Endocrinol.Metab.28,1763-1767).The existence of α subunit C-terminal halfcystine also can cause the heterodimer activity descend a little (table 3, Fig. 3), supposition effect consistent (Piece, the J.G.﹠amp of the contact of this part of this phenomenon and hormone performance acceptor; Parsons, T.F. (1981) Annu.Rev.Biochem.50,465-495; Chen, F., Wang, Y.﹠amp; Puett, D. (1992) Mol.Endocrinol.6,914-919).Yet the specific activity of these analogues lacks the analogue of these five carboxyl terminal α subunit residues strong a lot (Piece, J.G.﹠amp; Parsons, T.F. (1981) Annu.Rev.Biochem.50 465-495), points out any single residue in this zone unessential by hormonal activity, and each residue is only made the contribution of trace to hCG and LHR bonded total energy.Supposition based on the aspartic acid in α Lys91 and the membrane spaning domain contacts (Yoo, J., Ji, I.﹠amp; Ji, T.H. (1991) J.Biol.Chem.266,17741-17743), α Lys91 is essential (Ji, I., Zeng, H.﹠amp in the signal conduction; Ji, T.H. (1993) J.Biol.Chem.268,22971-22974), thus the heterodimer that contains α K91C to have kept most of energy (Fig. 3) of hCG just wondrous.HCG-α K91C kept this result of basic usefulness and this hypothesis inconsistent.Therefore prepared hCG-α K91E and hCG-α K91M and checked its activity.It is reported that these analogues have extremely low usefulness (Yoo, J., Ji, I.﹠amp; Ji, T.H. (1991) J.Biol.Chem.266,17741-17743).As shown in figure 17, although contain with the analogue avidity of the interactional glutaminate of LHR lowlyer 10 times than hCG, two analogues all have basic usefulness.This observed result with α Lys91 is changed into halfcystine gained observations (table 3 is consistent Fig. 3), point out this lysine residue as reporting for signal conduction not really important (Yoo, J., Ji, I.﹠amp; Ji, T.H. (1991) J.Biol.Chem.266,17741-17743).
Second ring of the C-terminal of β subunit and α subunit
In the conduction of receptors bind and signal is analyzed, in second ring of β subunit C-terminal and α subunit residue form have in the crosslinked sour stable analogs many have considerable activity (table 3, Fig. 4).These analogues comprise with second ring of α subunit on 35,37,40,44,42,50 and 52 analogues (table 3) that residue links to each other.Last 47 of second ring of β subunit C-terminal and α subunit links to each other its receptor-binding activity almost completely lost, and closes the active drop by half that makes heterodimer with 43 and 46 digit pairs.In the active analogue of tool, the side chain of second ring of α subunit residue is all outstanding to first ring of β subunit and the 3rd ring in nearly all analogue, and prompting is faced first ring of β subunit and do not contacted with LHR with second ring surface of α subunit of the 3rd ring.The side chain of the 43rd and the 46th residue of α subunit is outstanding to securing band, the side chain of 47 residues then and the little anchor ring of securing band that disulfide linkage forms between Cys93 and Cys100 right.Therefore, may destroy the interaction between these parts and acceptor on second ring of α subunit by the crosslinked loss of activity that causes of β subunit C-terminal and these sites, or change the conformation of second ring of α subunit in the mode that reduces this hormone and acceptor interaction ability.
138 residues of length prompting of β subunit C-terminal can cross first ring and the 3rd ring is connected in second residue (Fig. 1) on the ring.Therefore, any one β subunit C-terminal part all may not can occupy the ditch space of second ring of α subunit and first ring of β subunit and the 3rd interannular in these analogues.Be the activity of this part of research β subunit C-terminal, prepared the analogue of disappearance β subunit 11 6-135 position residue and disappearance β subunit 12 1-135 position residue in occupation of the analogue of this ditch.The β C-terminal of these analogues is too short, can not and second ring of α subunit on residue form crosslinked and do not cross ditch between this subunit.These analogues all have basic activity (Fig. 4) in the conduction of LHR and signal is analyzed, do not provide further support for this part of second ring of α subunit does not contact this viewpoint with LHR.
β subunit and first ring of α subunit and the 3rd ring
For learning that 8 residues of β subunit 13 whether can be partial cross-linked with other of α subunit core, hCG-β S138C residue on several first rings or the 3rd ring is expressed with halfcystine alternate α subunit analogs.Except contain the heterodimer that substitutes halfcystine in α Ser64 position, there only have small portion to take place in the gained heterodimer to be crosslinked, because they stability under condition of acidic pH lower (table 2).The analogue crosslinked with participating in subunit compared, have in the α subunit analogs of these heterodimers many halfcystines be positioned at apart from the securing band C-terminal far away many position, may exceed β subunit C-terminal district the scope (Fig. 1) that can effectively contact.In receptors bind and signaling activity analysis, α S64C/hCG-β S138C heterodimer has considerable activity (table 3), shows that this α subunit residue with LHR essential contacting does not take place.
Compare with arbitrary subunit generation halfcystine is alternative, crosslinked interaction to this heterodimer and LHR takes place and has bigger influence (Fig. 5) in β subunit C-terminal and α subunit carboxyl terminal residue.Prompting is as (Piece, J.G.﹠amp according to the hypothesis of doing with the institute before more than 25 years; Parsons, T.F. (1981) Annu.Rev.Biochem.50,465-495), α subunit C-terminal is near the contact surface of LH acceptor.Yet β subunit C-terminal is connected in this zone and may changes the structure of heterodimer or cause this end of β subunit to move on near other parts that this hormone contacts with acceptor.
Protein tubercle-hCG-beta lactamase
These researchs do not contact this idea a large amount of supports are provided for most of residue on second ring of α subunit does not participate in essential LH acceptor.But the possibility of this subregion of hormone near the acceptor contact do not got rid of in these researchs.For clarifying this problem, prepared size with the similar globular protein of hCG---beta lactamase is connected in the hCG β subunit analogs of specific position on the α subunit.Beta lactamase is merged producing hCG-β S138C-β LA140 and hCG-β S138C-β LA145 with 140 and 145 residues of hCG-β S138C respectively, thereby prepare these analogues.Back one analogue and the crosslinked halfcystine of α subunit and and the beta lactamase N-terminal take place to contain one " at interval " that comprises seven residues between the crosslinked halfcystine.With these β subunit analogs and α subunit analogs α T46C, α L48C, α S64C and α S92C co expression, the formation (Fig. 6) that causes acid to stablize heterodimer.This result shows that the existence of beta lactamase can not stop 8 residues of β subunit 13 to be connected on the α subunit.
Compare the activity of these hCG-beta lactamase analogues much lower (Fig. 6) with the corresponding analogs that does not contain beta lactamase.
Be designed for the front and differentiate that the α subunit does not participate in result of study (Xing, Y., Lin, W., Jiang, M., Myers, R.V., Cao, D., Bernard, the M.P.﹠amp of the part that key receptor contacts; Moyle, the prolan analogue that other modes of W.R. are folding: folding and bioactive prompting to hormone.Journal of Biological chemistry.2001.) integrate, these results expand at present the understanding to the surface that contact with LHR, and point out second ring of α subunit not participate in essential LH acceptor with the ditch of first ring of β subunit and the 3rd interannular to contact, and this contact is with requirement (Moyle, W.R., Campbell crucial in the previous model, R.K., Rao, S.N.V., Ayad, N.G., Bernard, M.P., Han, Y.﹠amp; Wang, Y. (1995) J.Biol.Chem.270,20020-20031.).Really, discern ability (Wang, Y.H., Bernard, the M.P.﹠amp of the hCG and the analogue thereof that are incorporated into LHR on the cell surface based on monoclonal antibody; Moyle, W.R. (2000) Mol.Cell.Endocrinol.170,67-77), and the binding ability of hCG/hFSH and hCG/hTSH mosaic and LHR [Campbell, R.K., Dean Emig, D.M.﹠amp; Moyle, W.R. (1991) Proc.Natl.Acad.Sci. (USA) 88,760-764; Campbell, R.K., Bergert, E.R., Wang, Y., Morris, J.C.﹠amp; Moyle, W.R. (1997) Nature Biotech.15,439-443; Grossmann, M., Szkudlinski, M.W., Wong, R., Dias, J.A., Ji, T.H.﹠amp; Weintraub, B.D. (1997) J.Biol.Chem.272,15532-15540; Moyle, W.R., Campbell, R.K., Myers, R.V., Bernard, M.P., Han, Y.﹠amp; Wang, X. (1994) Nature 368,251-255], as if seldom have the specific residue of hCG to participate in essential LHR contact.Those are positioned at securing band to the residue that the hCG activity has a significant impact, even but these residues change change (Han, Y., Bernard, the M.P.﹠amp of the interaction generation several times that also can not make hormone-acceptor one by one; Moyle, W.R. (1996) Mol.Cell.Endocrinol.124,151-161).The site of two apart from each others may influence hCG-LHR interaction (Bernard, M.P., Myers, R.V.﹠amp on the acceptor; Moyle, W.R. (1998) Biochem.J.335,611-617), conformational change small in while mammals metakentrin and this hormone of people LHR interaction partners is very responsive.Based on these observed results, we think as the interaction of tethelin and its acceptor, and the interaction of sugar-protein hormone and its acceptor also be can't help relative few contact and dominated [Clackson, T.﹠amp; Wells, J.A. (1995) Science 267,383-386; Wells, J.A. (1996) Proc.Natl.Acad.Sci. (USA) 83,1-6].
The cDNA of, coding hFSH β subunit corresponding with the mRNA translator unit is from the Christie Kelton of branch of Serono, Ares Advanced Technology, and 280 Pind Street, Randolph MA obtains.The β subunit aminoacid sequence that does not contain leading peptide is shown in (Fig. 8, SEQ IDNO:40).With PCR and cassette mutagenesis method hFSH is improved, thereby generated construct (Fig. 8, SEQ ID NO:41) with series system the 1st～108 of hFSH of coding and the 115th～145 residue of hCG, called after FC1-108 β.The FC1-108 beta amino acids sequence of being found in hFSH β subunit that does not contain leading peptide also as shown in Figure 8.Substitute the ShoI-Apal fragment of the construct of coding hCG-S138C β with the ShoI-Apal fragment of FC1-108 β, prepare the FC1-108 β analogue that Ser132 position residue is transformed into Cys with this, thereby generate FC1-108, S132C β (Fig. 8, SEQ ID NO:42).With FC1-108 β, the several of α subunit analogs shown in S132C β and Fig. 7 express in the COS-7 cell with method described in the embodiment 1.With antibody A 1 13 Hes
125The heterodimer content in the substratum is gone in the emiocytosis of I-B603 mensuration.Last antibody is the antibody of anti-α subunit, and back one antibody is and hFSH β subunit bonded monoclonal antibody.The hFSH of purifying is as standard substance.After acid treatment is dissociated noncrosslinking heterodimer, as indicated above with A113 and
125I-B603 redeterminates, to determine the content of cross-linked material in the sample.The crosslinked analogue of check gained stimulates the ability that cyclisation AMP gathers in the Chinese hamster ovary celI of expressing fsh receptor.
As shown in figure 11, the tubercle that is positioned at the 35th residue of α subunit only makes active slight decline of this FSH analogue.Tubercle is connected with the 92nd, 64,48,46,42,90,43,88 and 86 residue causes the activity of this analogue progressively to be lost.The distance that shows the 35th residue of α subunit and fsh receptor contact surface is not near, and other several residues seem near being subjected to contact level.Different with the interaction of LH acceptor with hCG, the existence of the 42nd and the 43rd residue superior thyroid tubercle plays stronger restraining effect to this FSH analogue and its receptors bind.This point shows, when FSH and hCG and its acceptor interacted respectively, the surface portion of second ring of α subunit and the distance of fsh receptor were than nearer with the distance of LH acceptor.86 residues of tubercle and two receptor alpha subunits link to each other this result show two parts this a part all near being subjected to contact level.
Embodiment 3---study the protein tubercle that difunctional hCG/hFSH mosaic butts up against LH and fsh receptor
With the chimeric cDNA sequence of ordinary method preparation coding, wherein the codon of hCG the 101st～114 amino acids is substituted by the corresponding section of its hFSH β subunit.This chimeric aminoacid sequence called after CFC101-114 β, Fig. 8 (SEQ ID NO:38) is depicted as the sequence of removing the leading peptide residue.Substitute the ShoI-ApaI fragment of the construct of coding hCG-S138C β with the ShoI-ApaI fragment of CFC101-114 β, prepare the CFC101-114 β analogue that Ser138 position residue is transformed into Cys with this, thereby generate CFC101-114 β, S132C (Fig. 8, SEQ ID NO:39).With CFC101-114 β, the several of α subunit analogs shown in S132C and Fig. 7 express in the COS-7 cell with method described in the embodiment 1.With antibody A 1 13 Hes
125The heterodimer content in the substratum is gone in the emiocytosis of I-B110 mensuration.Last antibody is the antibody of anti-α subunit, and back one antibody is and hCG β subunit bonded monoclonal antibody.The hCG of purifying is as standard substance.After acid treatment is dissociated noncrosslinking heterodimer, as described above with A113 and
125I-B110 redeterminates to determine the content of cross-linked material in the sample.The crosslinked analogue of check gained suppresses
125I-hCG and the Chinese hamster ovary celI bonded ability of expressing the LH acceptor, and suppress
125I-hFSH and the Chinese hamster ovary celI bonded ability (Figure 12) of expressing fsh receptor.The stimulation ability of also having checked it that cyclisation AMP in the Chinese hamster ovary celI of expressing fsh receptor and the Chinese hamster ovary celI of expressing fsh receptor is gathered simultaneously.
As shown in figure 12, compare respectively with the difunctional mosaic of the most of hCG β subunit C-terminal of disappearance, the tubercle that is positioned at the 35th residue of α subunit stops radio-labeling hCG or radio-labeling hFSH and LH or fsh receptor bonded ability can not cause interference to this mosaic.But the existence of the 37th residue superior thyroid tubercle of α subunit causes theatrical Different Results.This tubercle is very little to the ability influence of this mosaic and LH receptors bind, but itself and fsh receptor bonded ability are almost completely disappeared.The existence of the 43rd and the 46th superior thyroid tubercle of α subunit descends the binding ability of mosaic and two kinds of acceptors simultaneously.The existence of the 48th and the 52nd superior thyroid tubercle of α subunit is much smaller to the influence of mosaic and arbitrary receptor binding capacity.In embodiment 1 and embodiment 2 viewed, the existence of the 86th superior thyroid tubercle of α subunit makes mosaic all be subjected to severe inhibition with combining of two kinds of acceptors.The distance that shows contact surface between this part of hormone and mosaic and the two kinds of acceptors is all very near.The 91st observation that superior thyroid tubercle is done obtains similar result to the α subunit.
As Figure 13 and shown in Figure 14, nodular existence can make the activity of most of mosaic in LH acceptor and fsh receptor signal conduction mensuration descend.But the active degree that descends is different and different with being tried the acceptor that thing checks.For example, the existence of the 37th superior thyroid tubercle of the α subunit inhibition that makes fsh receptor excite cyclisation AMP to gather to be subjected to mediates cyclisation AMP than LH acceptor to gather the inhibition that is subjected to much better than.Generally speaking, tubercle is similar to relative influence and its ability that influences acceptor interaction of the ability of mosaic activated cell reaction.Like this, even metakentrin has similar structure with follicle stimulating hormone, even their acceptor is closely similar, and the mode of these parts and acceptor interaction is also inequality.Present embodiment has shown the ability of utilizing these probes discriminating protein-protein-protein interactions of the present invention.
Embodiment 4: the tubercle that contains β-Nei Xiananmei
Contain hCG β, the tubercle that the analogue of S138C β subunit has is relatively little, be made up of residue around the Ser138.These nodular sizes are enough to measure between part and its acceptor, relative less distance.But these tubercles are still too little, can not be used to detect the degree of closeness apart from the farther residue of acceptor.Can add that β-Nei Xiananmei makes probe become big, thereby can walk around this restriction.Selecting β-Nei Xiananmei is that its C-terminal and N-terminal are positioned at this proteinic surface simultaneously because its crystalline structure is known as probe.Like this, for generating at this hormone arbitrary terminal the fused protein of great use.Select for use another advantage of β-Nei Xiananmei to be, it is enzyme (Zlokarnik, G., Negulescu, P.A., Knapp, T.E., Mere, L, Burres, N., Feng, L., Whitney, M., Roemer, the K.﹠amp of a kind of high transformation ratio, cutting fluorogenic substrate; Tsien, R.Y. (1998) Science 279,84-88), this point when detecting fused protein of great use.According to this point, β-Nei Xiananmei is as effective reporter molecules, though differ widely (Moore, J.T., Davis, S.T.﹠amp of the purposes of place anticipation therewith; Dev, I.K. (1997) Anal.Biochem.247,203-209).β-Nei Xiananmei also can be suppressed (Strynadka, N.C., Jensen, S.E., Johns, K., Blanchard, H., Page, M., Matagne, A., Frere, J.M.﹠amp by bonded protein with it; James, M.N. (1994) Nature 368,657-660.).So just might the β-Nei Xiananmei tubercle further be increased by adding arrestin simply.
Prepare two hCG β that contain the β-Nei Xiananmei fused protein, the S138C derivative.One of them called after hCG β, S138C-β LA (long) (SEQ ID NO:44), or abbreviate length (LONG) as.As shown in figure 15, it contains the β with hCG, the β-Nei Xiananmei that the S138C C-terminal merges.With the PCR mutafacient system is that template prepares this protein with pUC18.Another probe called after hCG β, S138C-β LA (short) (SEQ ID NO:43), or abbreviate weak point (SHORT) as.As shown in figure 15, it contains the β with brachymemma hCG, the β-Nei Xiananmei that S138C merges.Short protein only has an amino acid between coupling halfcystine and β-Nei Xiananmei zero position.Clearly, the residue that inserts different quantities between coupling halfcystine and β-Nei Xiananmei can prepare this proteinoid of different editions.While, it was halfcystine institute alternate β-Nei Xiananmei analogue that the those of skill in the art in molecular simulation and the biology field can design and prepare a surface residue admittedly.This halfcystine can serve as the coupling halfcystine, this point can make the β-Nei Xiananmei tubercle with than with afterbody more inflexible be connected and fixed on the target halfcystine.
Generate five differences that contain long and short β subunit and stablize heterodimer.Preparing these dimers is in order to measure the degree of closeness of the 46th, 48,52,64 and 92 residues of α subunit with respect to the LH acceptor.As Figure 16 and shown in Figure 17, these analogue nones have the activity suitable with hCG.Long and short β subunit probe and the 52nd residue link coupled heterodimer of α subunit [promptly are respectively α N52C+hCG β, S138C-β LA (long) and N52C+hCG β, S138C-β LA (short)] in measuring, the signal conduction kept the major part of hCG active.α subunit Asn52 residue is become halfcystine the glycosylation signal is destroyed, cause the oligosaccharides on second ring of α subunit to be lost.This oligosaccharides is lost from hCG and is caused its usefulness nearly 60% (Figure 18) that descend.Like this, connect the β-Nei Xiananmei tubercle in this site and can cause second oligosaccharides on the ring to be lost, thereby make its usefulness forfeiture.These long and short analogues all have higher relatively activity, α subunit residue is described and keeps clear of the acceptor contact surface.Since it is high-effect that two analogues all have, these results suggest oligosaccharides are played a role in the signal conduction of hCG mediation.The effect of oligosaccharides is the position of upsetting subunit in the hCG on second ring of activity prompting α subunit of long and short analogue, and this point does not rely on specific contact the between oligosaccharides and arbitrary subunit.
Long and short analogue activity is still kept, and illustrates that the 48th of α subunit and 64 residues are than the also close acceptor of being measured by the activity of hCG β S18C+ α L48C and hCG β S138C+ α S64C.This discovery has significant effects to hCG-LH receptor complex model.Also proved conclusively of the early discovery of the 46th and 92 residues of α subunit near receptor surface.
Embodiment 5: α subunit N-terminal has the tubercle of a coupling halfcystine
The coupling halfcystine is not to be positioned at proteinic C-terminal, and we have designed the analogue that the coupling halfcystine is positioned at the protein amino end.(β 101-145, aminoacid sequence α) is as follows for such analogue.The preparation of this analogue comprises the residue of the 3rd～100 β subunit of deletion hCG, uses conventional PCR and cassette mutagenesis method that the end of α subunit and gained β subunit is merged.This analogue contains halfcystine freely at the 12nd residue place, can serve as the coupling halfcystine.
10????????20????????30????????40????????50????????60
β101-145，α(SEQ?ID?NO：54)
skggpkdhpltcddprfqdsssskapppslpspsrlpgpsdtpilpqapdvqdcpectlq-
70????????80????????90???????100???????110???????120
enpffsqpgapllqcmgccfsrayptplrskktmlvqknvtsestccvaksynrvtvmgg-
130???????140
fkvenhtachcstcyyhks
Embodiment 6---the nodular generation method of site-specific protein
The protein of selecting tubercle to connect.Then, determine to want on this protein the specific site of mark.Can express this proteinic construct with mutafacient system preparation known in the art, the natural residue at second protein specific markers place is substituted by halfcystine.In addition, encoded protein matter also comprises afterbody that contains a halfcystine that is connected with protein one end and the tubercle that is connected in tail end in the construct.Then construct is inserted express cell, be connected with nodular protein thereby generate on the specific site.
On protein, add tubercle, comprise: tubercle need be located part and be introduced halfcystine from the teeth outwards, and at proteinic N-terminal or C-terminal position introducing halfcystine, so that the halfcystine of protein terminal can form disulfide linkage with the added halfcystine in surface.The formation of disulfide linkage will make the protein stabilization that contains halfcystine in produce nodular surperficial (see figure 9) in this site between these two halfcystines.
In order to generate tubercle, the protein terminal that contains halfcystine needs sufficiently long, makes its halfcystine form disulfide linkage with the halfcystine that is about to comprise on this nodular site.This may need to add an afterbody such as hCG β subunit C-terminal, and hFSH is added the nodular situation of protein is identical.The composition of afterbody can alter a great deal, and is not limited to hCG β subunit C-terminal.The afterbody main requirement is: its length should be enough to and can interact with the halfcystine of definite nodule position on the protein surface, does not contain simultaneously to stop it to arrive the residue in this site.These residues comprise the characteristic residue of membrane spaning domain, and the residue in generation and other protein or wherein a part of bonded site, and wherein the halfcystine on these protein and the tubercle place protein surface is far apart.
The residue of any side of halfcystine can form the tubercle (see figure 9) on protein amino end or the C-terminal.Usually tubercle is also big more more at most for this site residue number.Minimum tubercle may only contain a halfcystine (Figure 10 A).The nodular generation method of this class is included in the end of connector N-terminal or C-terminal and introduces a halfcystine, and next-door neighbour's halfcystine part is introduced cleavage site (seeing Figure 10 A).Cleavage site should contain one section other part of this protein do not find, can be by the aminoacid sequence of protease hydrolysis.At least can make tubercle become big by three kinds of modes.First kind of mode is that a kind of protein and this nodular protein of structure are merged (seeing Figure 10 B) endways.As previously mentioned, β-Nei Xiananmei and protein terminal are merged.But β-Nei Xiananmei is not the unique protein that is fit to this purpose.Selecting β-Nei Xiananmei is that its crystalline structure shows that its N-terminal is positioned at the surface as the convenience of probe, and such position is beneficial to construction of fusion protein matter.The second way is with proteolytic enzyme connector to be cut, and reduces the size of connector and to nodular influence.The 3rd, by increasing or reduce amino acid number, change the distance between coupling halfcystine and fused protein probe.Seen in hCG embodiment, the distance shortening has limited nodular moving, and the activity of the short more then hormone of distance descends serious more.This point makes tubercle remain on the position that is subjected to contact level near hormone and LH.
As among Figure 10 obviously as seen, the coupling halfcystine is not to be positioned at afterbody.On fused protein surface as a tubercle part, can form the disulfide linkage part with target halfcystine on the protein and introduce halfcystine, on the protein surface that this fused protein will be directly connected in need to survey (seeing Figure 10 C).This method can be used for controlling as the fused protein of a tubercle part towards.When proteolytic enzyme is used as fused protein, introduces halfcystine on distance reactive site surface far away and can make the reactive site of fused protein keep clear of the surface that tubercle will connect.
Embodiment 7: the afterbody of using brachymemma is as probe
Add the distance that tubercle can be measured two protein surfaces toward protein.As shown, second of the α subunit ring do not contact with acceptor basically.The coupling halfcystine at hCG β subunit the 138th amino acids place location is less relatively to the constraint of the position that can be occupied by β subunit 111-137 position residue, and wherein 111-137 position residue links to each other the core of the coupling halfcystine on the tubercle with hCG β subunit.Afterbody itself also can be used for surveying proteinic surface, has several with the example of its position constraint in the molecule specific position.These examples comprise that the part with the protein active position covers, thereby make it not possess activity before this afterbody is fallen by proteolytic cleavage always.
In the research of hCG-LH acceptor interaction, contact this and assert in order to check second ring of α subunit and the ditch of first ring of β subunit and the 3rd interannular to form crucial acceptor, afterbody is limited in will be comparatively desirable in this ditch.Act on aforesaid afterbody and can make its this part of passing hormone, but not necessarily force to make it be positioned this position.Because its length, afterbody may be crossed the convex surface of first ring of β subunit and the 3rd ring nodular coupling halfcystine can be contacted by different halfcystines with alternate on second ring of α subunit.Therefore, when 42,46 or 48 residues substituted halfcystines and are target spot on second ring of α subunit, we forced the afterbody brachymemma to make it cross the ditch of second ring of α subunit and first ring of β subunit and the 3rd interannular.Afterbody comparison thing hCG β, δ 116-135, S138C (SEQ ID NO:45) and hCG β, δ 121-135, S138C (SEQ ID NO:46) length (Figure 15) is too short, can not make between tubercle and target protein and form disulfide linkage, unless afterbody crosses the ditch of second ring of α subunit and first ring of β subunit and the 3rd interannular.As Figure 23 and Figure 24 finding as a result, contain α T46C and hCG β, δ 116-135, S138C or hCG β, δ 121-135, the heterodimer of S138C has basic receptor-binding activity.Contain α L48C and hCG β, δ 121-135, the heterodimer of S138C also has appreciable receptor-binding activity.Less α L48C and the hCG β of containing arranged, δ 121-135, S138C, acid stablize heterodimer and are formed, and point out the short-tail portion that is tried may have the sufficient length that tubercle coupling halfcystine is contacted with target halfcystine on the protein.Since the afterbody of analogue not necessarily crossed the ditch of second ring of α subunit and first ring of β subunit and the 3rd interannular when tubercle coupling halfcystine contacted with the target halfcystine at residue place, α subunit 42 position, select this analogue as positive control.
In sum, these observed results illustrate that this ditch does not participate in essential acceptor contact.Position how to handle afterbody also has been described so that it is near proteinic specific part simultaneously.This point can make afterbody be used to shield specific site, and this characteristic and the cleavage site coupling of having introduced afterbody will be very useful when the proteolytic enzyme of preparation latent form, toxin or other useful analogues.The afterbody in shielding proteolytic enzyme or detoxifying function site can be used to prepare and use the reagent that can enter cell, and these reagent can be cut afterbody by endogenic or other lytic enzymes after entering cell, and toxin is activated.These reagent will be very useful when treatment tumour or other diseases.
It is said that the little ring in the securing band plays a role when hCG brings into play its biologic activity, need connect tubercle in this part of β subunit for studying this possibility.Preparation contains the α subunit analogs of hCG β subunit carboxyl terminal residue.Although shown that the part of β subunit C-terminal is received α subunit C-terminal can make the activity of heterodimer descend 50 times even more, uses complete β subunit sequence can not cause such consequence.The analogue that contains the complete C-terminal sequence of hCG β subunit that is connected in the α subunit, comprising aminoacid sequence Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln, is hCG active 50% or more in receptors bind and its activity of signal conduction mensuration.We find that this may be for having near the part due to the charged residue existence being connected of α subunit source residue and β subunit source residue.On the β subunit residue that tubercle is connected in the little ring of securing band, the end of the hCG β subunit C-terminal of brachymemma being received the α subunit is to generate hCG-α CT δ 116/135, S138C (SEQ ID NO:57).With itself and Arg94 (SEQ ID NO:58), Arg95 (SEQ ID NO:59), Ser96 (SEQ ID NO:60), Thr97 (SEQ ID NO:61), Thr98 (SEQ ID NO:62) and Asp99 (SEQ ID NO:63) by halfcystine alternate β subunit analogs coexpression in the COS-7 cell.From Figure 26,27,28,29,30,31 and the data of Figure 32 as can be known, these protein all form the stable heterodimer of acid, how this part that these researchs have disclosed the β subunit interacts with LH acceptor (LHR).The existence of the 95th and 99 residue superior thyroid tubercles interacts its LHR and biologic activity disappears.When tubercle was positioned on the 96th or 97 residues, this nodular influence was much smaller because these analogues in conjunction with and the signal conduction have basic activity in measuring.When tubercle was connected in the 98th residue, tubercle further weakened the interaction of acceptor, but was not attenuated to the degree when being connected in the 95th and 99 residues.The prompting latter may be positioned near being subjected to the contact level part.
The 95th residue side chain of hCG β subunit towards with the 94th and 95 residue side chain towards opposite, this phenomenon possible explanation connects tubercle than connecting tubercle and more can suppress hCG-LHR on the 94th and 96 residue and interact this fact in this site.This some prompting near the securing band surface of Arg95 side chain may be subjected to contact level approaching.For learning whether this side chain contacts with proteinic surface, preparation contains more nodular analogue.The construction process of these analogues comprises that the afterbody that maybe will contain Gly-Gly-Cys is linked the C-terminal of α subunit (SEQ ID NO:36) with alternative the 92nd Serine of α subunit (SEQ ID NO:35) of halfcystine.From Figure 30 and Figure 32 as can be known, littler tubercle disturbs the active ability of heterodimer much lower in LHR measures.Like this, two stable crosslinked heterodimers of acid have all kept appreciable activity in these are measured.Illustrate that the side chain of β subunit Arg95 residue is very possible near being subjected to contact level, but be not that acceptor contact institute is essential.The close little ring of securing band in position of the C-terminal of hormone and LHR interaction back α subunit also is described simultaneously.
Since it is the same with the oligosaccharides at second ring of α subunit Asn52 place, the C-terminal of α subunit is by the sugar-protein hormonal activity is essential completely, and how the change of understanding α subunit C-terminal position in heterodimer influences its biologic activity will be very interesting.Preparation because of Asn52 changes Asp into can not glycosylation on second ring of α subunit, can add the α subunit analogs (SEQ ID NO:65) of halfcystine tubercle at the 92nd, 94,95 and 96 residues of β subunit.With this analogue and the β subunit analogs coexpression in the COS-7 cell that contains alternative halfcystine at the 92nd, 94,95 and 96 residue place, make it to produce the stable crosslinked heterodimer of acid.These analogues reach its activity of chimeric β subunit analogs (SEQ ID NO:66) that contains alternative halfcystine at the Arg96 place and show that the C-terminal of α subunit can be connected with several sites in the β subunit, points out its position suitably not fix (Figure 33).And low this result of the activity of chimeric analogs shows that this a part of conformation of hormone is influential to the signal conduction.
The present invention is very suitable for implementing, and that acquisition is mentioned and inherent net result and benefit, this point are to understand easily to the one skilled in the art.Method described here and operation go on foot poly-and composition is the representative of present preferred embodiment, are exemplary, scope of the present invention are not made restriction.Those of skill in the art can change it or are used for other purposes around spirit of the present invention or scope that claims are decided in this area.
Do not deviate from scope of the present invention and spirit and the present invention disclosed herein is carried out various to substitute and modify for those of skill in the art in this area be conspicuous.
Point out specifically, individually that as each publication patent or patent application are hereby incorporated by in the same way so that its integral body is incorporated herein by reference.Following is the reference that possible be incorporated herein by reference.
Reference:
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Sequence table
<110>Moyle，William?R.
Xing，Yongna
＜120〉protein tubercle
<130>268/279-RWJ-01-40
<140>60/345,283
<141>2001-11-08
<160>56
<170>PatentIn?version?3.1
<210>1
<211>92
<212>PRT
<213>Homo?sapiens
<400>1
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>2
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Gln5
<400>2
Ala?Pro?Asp?Val?Cys?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>3
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Leu12
<400>3
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Cys?Gln?Glu?Asn?Pro
1???????????????5????????????????????10?????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>4
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Asn15
<400>4
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Cys?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>5
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Phe17
<400>5
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Cys?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65???????????????????70?????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>6
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Leu22
<400>6
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Cys?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>7
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Gln7
<400>7
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Cys?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>8
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?subsituted?for?Leu22
<400>8
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Cys?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>9
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?subsituted?for?Arg35
<400>9
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Cys?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>10
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Tyr37
<400>10
Ala?Pro?Asp?Va1?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Mst?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Cys?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>11
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Pro38
<400>11
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Cys?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>12
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Thr39
<400>12
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Cys?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>13
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Pro40
<400>13
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Cys?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>14
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Leu41
<400>14
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Cys?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75?????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>15
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Arg42
<400>15
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Cys?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>16
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Ser43
<400>16
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cye?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Cys?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>17
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Lys44
<400>17
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Cys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>18
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Lys45
<400>18
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Cys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>19
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha?subunit?with?Cys?substituted?for?Thr46
<400>19
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Cys?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>20
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Met47
<400>20
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Cys?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>21
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Leu48
<400>21
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Pra?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Cys
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????70??????????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????????90
<210>22
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Val49
<400>22
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Cys?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>23
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Gln50
<400>23
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Cys?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>24
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Lys51
<400>24
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Cys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>25
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Asn52
<400>25
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Cys?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>26
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Val53
<400>26
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Cys?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>27
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Glu56
<400>27
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Cys?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>28
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Ser64
<400>28
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Cys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>29
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Val76
<400>29
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Cys?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>30
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Thr86
<400>30
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Cys?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>31
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Tyr88
<400>31
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Cys?Tyr?His?Lys?Ser
85??????????????????90
<210>32
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Leu89
<400>32
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Cys?His?Lys?Ser
85??????????????????90
<210>33
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?His90
<400>33
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lye?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?Cys?Lys?Ser
85??????????????????90
<210>34
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Lys91
<400>34
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Cys?Ser
85??????????????????90
<210>35
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?with?Cys?substituted?for?Ser92
<400>35
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Cys
85??????????????????90
<210>36
<211>145
<212>PRT
<213>Homo?sapiens
<400>36
Ser?Lys?Glu?Pro?Leu?Arg?Pro?Arg?Cys?Arg?Pro?Ile?Asn?Ala?Thr?Leu
1???????????????5???????????????????10??????????????????15
Ala?Val?Glu?Lys?Glu?Gly?Cys?Pro?Val?Cys?Ile?Thr?Val?Asn?Thr?Thr
20??????????????????25??????????????????30
Ile?Cys?Ala?Gly?Tyr?Cys?Pro?Thr?Met?Thr?Arg?Val?Leu?Gln?Gly?Val
35??????????????????40??????????????????45
Leu?Pro?Ala?Leu?Pro?Gln?Val?Val?Cys?Asn?Tyr?Arg?Asp?Val?Arg?Phe
50??????????????????55??????????????????60
Glu?Ser?Ile?Arg?Leu?Pro?Gly?Cys?Pro?Arg?Gly?Val?Asn?Pro?Val?Val
65??????????????????70??????????????????75??????????????????80
Ser?Tyr?Ala?Val?Ala?Leu?Ser?Cys?Gln?Cys?Ala?Leu?Cys?Arg?Arg?Ser
85??????????????????90??????????????????95
Thr?Thr?Asp?Cys?Gly?Gly?Pro?Lys?Asp?His?Pro?Leu?Thr?Cys?Asp?Asp
100?????????????????105?????????????????110
Pro?Arg?Phe?Gln?Asp?Ser?Ser?Ser?Ser?Lys?Ala?Pro?Pro?Pro?Ser?Leu
115?????????????????120?????????????????125
Pro?Ser?Pro?Ser?Arg?Leu?Pro?Gly?Pro?Ser?Asp?Thr?Pro?Ile?Leu?Pro?Gln
130?????????????????135?????????????????140
<210>37
<211>145
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?beta-subunit?with?Cys?substituted?for?Ser138
<400>37
Ser?Lys?Glu?Pro?Leu?Arg?Pro?Arg?Cys?Arg?Pro?Ile?Asn?Ala?Thr?Leu
1???????????????5???????????????????10??????????????????15
Ala?Val?Glu?Lys?Glu?Gly?Cys?Pro?Val?Cys?Ile?Thr?Val?Asn?Thr?Thr
20??????????????????25??????????????????30
Ile?Cys?Ala?Gly?Tyr?Cys?Pro?Thr?Met?Thr?Arg?Val?Leu?Gln?Gly?Val
35??????????????????40??????????????????45
Leu?Pro?Ala?Leu?Pro?Gln?Val?Val?Cys?Asn?Tyr?Arg?Asp?Val?Arg?Phe
50??????????????????55??????????????????60
Glu?Ser?Ile?Arg?Leu?Pro?Gly?Cys?Pro?Arg?Gly?Val?Asn?Pro?Val?Val
65??????????????????70??????????????????75??????????????????80
Ser?Tyr?Ala?Val?Ala?Leu?Ser?Cys?Gln?Cys?Ala?Leu?Cys?Arg?Arg?Ser
85??????????????????90??????????????????95
Thr?Thr?Asp?Cys?Gly?Gly?Pro?Lys?Asp?His?Pro?Leu?Thr?Cys?Asp?Asp
100?????????????????105?????????????????110
Pro?Arg?Phe?Gln?Asp?Ser?Ser?Ser?Ser?Lys?Ala?Pro?Pro?Pro?Ser?Leu
115?????????????????120?????????????????125
Pro?Ser?Pro?Ser?Arg?Leu?Pro?Gly?Pro?Cys?Asp?Thr?Pro?Ile?Leu?Pro?Gln
130?????????????????135?????????????????140
<210>38
<211>145
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?beta-subunit?residues?101-114?were?replaced?with?their?hFSH?b
eta-subunit?counterparts，namely?hFSH?beta-subunit?residues?95-10
8
<400>38
Ser?Lys?Glu?Pro?Leu?Arg?Pro?Arg?Cys?Arg?Pro?Ile?Asn?Ala?Thr?Leu
1???????????????5???????????????????10??????????????????15
Ala?Val?Glu?Lys?Glu?Gly?Cys?Pro?Val?Cys?Ile?Thr?Val?Asn?Thr?Thr
20??????????????????25??????????????????30
Ile?Cys?Ala?Gly?Tyr?Cys?Pro?Thr?Met?Thr?Arg?Val?Leu?Gln?Gly?Val
35??????????????????40??????????????????45
Leu?Pro?Ala?Leu?Pro?Gln?Val?Val?Cys?Asn?Tyr?Arg?Asp?Val?Arg?Phe
50??????????????????55??????????????????60
Glu?Ser?Ile?Arg?Leu?Pro?Gly?Cys?Pro?Arg?Gly?Val?Pro?Asn?Val?Val
65??????????????????70??????????????????75??????????????????80
Ser?Tyr?Ala?Val?Ala?Leu?Ser?Cys?Gln?Cys?Ala?Leu?Cys?Arg?Arg?Ser
85??????????????????90??????????????????95
Thr?Thr?Asp?Cys?Thr?Val?Arg?Gly?Leu?Gly?Pro?Ser?Tyr?Cys?Ser?Phe
100?????????????????105?????????????????110
Gly?Glu?Phe?Gln?Asp?Ser?Ser?Ser?Ser?Lys?Ala?Pro?Pro?Pro?Ser?Leu
115?????????????????120?????????????????125
Pro?Ser?Pro?Ser?Arg?Leu?Pro?Gly?Pro?Ser?Asp?Thr?Pro?Ile?Leu?Pro?Gln
130?????????????????135?????????????????140
<210>39
<211>145
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?beta-subunit?residues?101-114?were?replaced?with?their?hFSH?b
eta-subunit?counterparts，namely?hFSH?beta-subunit?residues?95-10
8，and?Serine38?in?the?beta-subunit?carboxyterminus?of?this
analog?was?replaced?with?Cys
<400>39
Ser?Lys?Glu?Pro?Leu?Arg?Pro?Arg?Cys?Arg?Pro?Ile?Asn?Ala?Thr?Leu
1???????????????5???????????????????10??????????????????15
Ala?Val?Glu?Lys?Glu?Gly?Cys?Pro?Val?Cys?Ile?Thr?Val?Asn?Thr?Thr
20??????????????????25??????????????????30
Ile?Cys?Ala?Gly?Tyr?Cys?Pro?Thr?Met?Thr?Arg?Val?Leu?Gln?Gly?Val
35??????????????????40??????????????????45
Leu?Pro?Ala?Leu?Pro?Gln?Val?Val?Cys?Asn?Tyr?Arg?Asp?Val?Arg?Phe
50??????????????????55??????????????????60
Glu?Ser?Ile?Arg?Leu?Pro?Gly?Cys?Pro?Arg?Gly?Val?Pro?Asn?Val?Val
65??????????????????70??????????????????75??????????????????80
Ser?Tyr?Ala?Val?Ala?Leu?Ser?Cys?Gln?Cys?Ala?Leu?Cys?Arg?Arg?Ser
85??????????????????90??????????????????95
Thr?Thr?Asp?Cys?Thr?Val?Arg?Gly?Leu?Gly?Pro?Ser?Tyr?Cys?Ser?Phe
100?????????????????105?????????????????110
Gly?Glu?Phe?Gln?Asp?Ser?Ser?Ser?Ser?Lys?Ala?Pro?Pro?Pro?Ser?Leu
115?????????????????120?????????????????125
Pro?Ser?Pro?Ser?Arg?Leu?Pro?Gly?Pro?Cys?Asp?Thr?Pro?Ile?Leu?Pro?Gln
130?????????????????135?????????????????140
<210>40
<211>111
<212>PRT
<213>Homos?sapiens
<400>40
Asn?Ser?Cys?Glu?Leu?Thr?Asn?Ile?Thr?Ile?Ala?Val?Glu?Lys?Glu?Gly
1???????????????5???????????????????10??????????????????15
Cys?Gly?Phe?Cys?Ile?Thr?Ile?Asn?Thr?Thr?Trp?Cys?Ala?Gly?Tyr?Cys
20??????????????????25??????????????????30
Tyr?Thr?Arg?Asp?Leu?Val?Tyr?Lys?Asp?Pro?Ala?Arg?Pro?Lys?Ile?Gln
35??????????????????40??????????????????45
Lys?Thr?Cys?Thr?Phe?Lys?Glu?Leu?Val?Tyr?Glu?Thr?Val?Arg?Val?Pro
50??????????????????55??????????????????60
Gly?Cys?Ala?His?His?Ala?Asp?Ser?Leu?Tyr?Thr?Tyr?Pro?Val?Ala?Thr
65??????????????????70??????????????????75??????????????????80
Gln?Cys?His?Cys?Gly?Lys?Cys?Asp?Ser?Asp?Ser?Thr?Asp?Cys?Thr?Val
85??????????????????90??????????????????95
Arg?Gly?Leu?Gly?Pro?Ser?Tyr?Cys?Ser?Phe?Gly?Glu?Met?Lys?Glu
100?????????????????105?????????????????110
<210>41
<211>139
<212>PRT
<213>Artificial?Sequence
<220>
<223>hFSH?beta-subunit?analog?lacking?the?leader?peptide?of?hFSH?beta-
subunit?with?hFSH?residues?1-108?and?hCG?residues?115-145?in
tandem
<400>41
Asn?Ser?Cys?Glu?Leu?Thr?Asn?Ile?Thr?Ile?Ala?Val?Glu?Lys?Glu?Gly
1???????????????5???????????????????10??????????????????15
Cys?Gly?Phe?Cys?Ile?Thr?Ile?Asn?Thr?Thr?Trp?Cys?Ala?Gly?Tyr?Cys
20??????????????????25??????????????????30
Tyr?Thr?Arg?Asp?Leu?Val?Tyr?Lys?Asp?Pro?Ala?Arg?Pro?Lys?Ile?Gln
35??????????????????40??????????????????45
Lys?Thr?Cys?Thr?Phe?Lys?Glu?Leu?Val?Tyr?Glu?Thr?Val?Arg?Val?Pro
50??????????????????55??????????????????60
Gly?Cys?Ala?His?His?Ala?Asp?Ser?Leu?Tyr?Thr?Tyr?Pro?Val?Ala?Thr
65??????????????????70??????????????????75??????????????????80
Gln?Cys?His?Cys?Gly?Lys?Cys?Asp?Ser?Asp?Ser?Thr?Asp?Cys?Thr?Val
85??????????????????90??????????????????95
Arg?Gly?Leu?Gly?Pro?Ser?Tyr?Cys?Ser?Phe?Gly?Glu?Phe?Gln?Asp?Ser
100?????????????????105?????????????????110
Ser?Ser?Ser?Lys?Ala?Pro?Pro?Pro?Ser?Leu?Pro?Ser?Pro?Ser?Arg?Leu
115?????????????????120?????????????????125
Pro?Gly?Pro?Ser?Asp?Thr?Pro?Ile?Leu?Pro?Gln
130?????????????????135
<210>42
<211>137
<212>PRT
<213>Artificial?Sequence
<220>
<223>hFSH?beta-subunit?analog?lacking?the?leader?peptide?of?hFSH?beta-
subunit?with?hFSH?residues?1-108?and?hCG?residues?115-145?in?tand
em?and?with?Ser132?replaced?with?Cys
<400>42
Asn?Ser?Cys?Glu?Leu?Thr?Asn?Ile?Thr?Ile?Ala?Val?Glu?Lys?Glu?Gly
1???????????????5???????????????????10??????????????????15
Cys?Gly?Phe?Cys?Ile?Thr?Ile?Asn?Thr?Thr?Trp?Cys?Ala?Gly?Tyr?Cys
20??????????????????25??????????????????30
Tyr?Thr?Arg?Asp?Leu?Val?Tyr?Lys?Asp?Pro?Ala?Arg?Pro?Lys?Ile?Gln
35??????????????????40??????????????????45
Lys?Thr?Cys?Thr?Phe?Lys?Glu?Leu?Val?Tyr?Glu?Thr?Val?Arg?Val?Pro
50??????????????????55??????????????????60
Gly?Cys?Ala?His?His?Ala?Asp?Ser?Leu?Tyr?Thr?Tyr?Pro?Val?Ala?Thr
65??????????????????70??????????????????75??????????????????80
Gln?Cys?His?Cys?Gly?Lys?Cys?Asp?Ser?Asp?Ser?Thr?Asp?Cys?Thr?Val
85??????????????????90??????????????????95
Arg?Gly?Leu?Gly?Pro?Ser?Tyr?Cys?Ser?Phe?Gly?Glu?Phe?Gln?Asp?Ser
100?????????????????105?????????????????110
Ser?Ser?Ser?Lys?Ala?Pro?Pro?Pro?Ser?Leu?Pro?Ser?Pro?Ser?Arg?Leu
115?????????????????120?????????????????125
Pro?Gly?Pro?Cys?Asp?Thr?Pro?Ile?Leu
130?????????????????135
<210>43
<211>401
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCGbeta，S138C-betaLA(short)，beta-lactamase?fused?to?a?truncated
version?of?hCGbeta，S138C
<400>43
Ser?Lys?Glu?Pro?Leu?Arg?Pro?Arg?Cys?Arg?Pro?Ile?Asn?Ala?Thr?Leu
1???????????????5???????????????????10??????????????????15
Ala?Val?Glu?Lys?Glu?Gly?Cys?Pro?Val?Cys?Ile?Thr?Val?Asn?Thr?Thr
20??????????????????25??????????????????30
Ile?Cys?Ala?Gly?Tyr?Cys?Pro?Thr?Met?Thr?Arg?Val?Leu?Gln?Gly?Val
35??????????????????40??????????????????45
Leu?Pro?Ala?Leu?Pro?Gln?Val?Val?Cys?Asn?Tyr?Arg?Asp?Val?Arg?Phe
50??????????????????55??????????????????60
Glu?Ser?Ile?Arg?Leu?Pro?Gly?Cys?Pro?Arg?Gly?Val?Asn?Pro?Val?Val
65??????????????????70??????????????????75??????????????????80
ser?Tyr?Ala?Val?Ala?Leu?Ser?Cys?Gln?Cys?Ala?Leu?Cys?Arg?Arg?Ser
85??????????????????90??????????????????95
Thr?Thr?Asp?Cys?Gly?Gly?Pro?Lys?Asp?His?Pro?Leu?Thr?Cys?Asp?Asp
100?????????????????105?????????????????110
Pro?Arg?Phe?Gln?Asp?Ser?Ser?Ser?Ser?Lys?Ala?Pro?Pro?Pro?Ser?Leu
115?????????????????120?????????????????125
Pro?Ser?Pro?Ser?Arg?Leu?Pro?Gly?Pro?Cys?Asp?His?Pro?Glu?Thr?Leu
130?????????????????135?????????????????140
Val?Lys?Val?Lys?Asp?Ala?Glu?Asp?Gln?Leu?Gly?Ala?Arg?Val?Gly?Tyr
145?????????????????150?????????????????155?????????????????160
Ile?Glu?Leu?Asp?Leu?Asn?Ser?Gly?Lys?Ile?Leu?Glu?Ser?Phe?Arg?Pro
165?????????????????170?????????????????175
Glu?Glu?Arg?Phe?Pro?Met?Met?Ser?Thr?Phe?Lys?Val?Leu?Leu?Cys?Gly
180?????????????????185?????????????????190
Ala?Val?Leu?Ser?Arg?Ile?Asp?Ala?Gly?Gln?Glu?Gln?Leu?Gly?Arg?Arg
195?????????????????200?????????????????205
Ile?His?Tyr?Ser?Gln?Asn?Asp?Leu?Val?Glu?Tyr?Ser?Pro?Val?Thr?Glu
210?????????????????215?????????????????220
Lys?His?Leu?Thr?Asp?Gly?Met?Thr?Val?Arg?Glu?Leu?Cys?Ser?Ala?Ala
225?????????????????230?????????????????235?????????????????240
Ile?Thr?Met?Ser?Asp?Asn?Thr?Ala?Ala?Asn?Leu?Leu?Leu?Thr?Thr?Ile
245?????????????????250?????????????????255
Gly?Gly?Pro?Lys?Glu?Leu?Thr?Ala?Phe?Leu?His?Asn?Met?Gly?Asp?His
260?????????????????265?????????????????270
Val?Thr?Arg?Leu?Asp?Arg?Trp?Glu?Pro?Glu?Leu?Asn?Glu?Ala?Ile?Pro
275?????????????????280?????????????????285
Asn?Glu?Arg?Asp?Thr?Thr?Met?Pro?Val?Ala?Met?Ala?Thr?Thr?Leu?Arg
290?????????????????295?????????????????300
Lys?Leu?Leu?Thr?Gly?Glu?Leu?Leu?Thr?Leu?Ala?Ser?Arg?Gln?Gln?Leu
305?????????????????310?????????????????315?????????????????320
Ile?Asp?Trp?Met?Glu?Ala?Asp?Lys?Val?Ala?Gly?Pro?Leu?Leu?Arg?Ser
325?????????????????330?????????????????335
Ala?Leu?Pro?Ala?Gly?Trp?Phe?Ile?Ala?Asp?Lys?Ser?Gly?Ala?Gly?Glu
340?????????????????345?????????????????350
Arg?Gly?Ser?Arg?Gly?Ile?Ile?Ala?Ala?Leu?Gly?Pro?Asp?Gly?Lys?Pro
355?????????????????360?????????????????365
Ser?Arg?Ile?Val?Val?Ile?Tyr?Thr?Thr?Gly?Ser?Gln?Ala?Thr?Met?Asp
370?????????????????375?????????????????380
Glu?Arg?Asn?Arg?Gln?Ile?Ala?Glu?Ile?Gly?Ala?Ser?Leu?Ile?Lys?His
385?????????????????390?????????????????395?????????????????400
Trp
<210>44
<211>408
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCGbeta，S138C-betaLA(long)，beta-lactamase?fused?to?the?carboxyte
rminal?end?of?hCGb，S138C
<400>44
Ser?Lys?Glu?Pro?Leu?Arg?Pro?Arg?Cys?Arg?Pro?Ile?Asn?Ala?Thr?Leu
1???????????????5???????????????????10??????????????????15
Ala?Val?Glu?Lys?Glu?Gly?Cys?Pro?Val?Cys?Ile?Thr?Val?Asn?Thr?Thr
20??????????????????25??????????????????30
Ile?Cys?Ala?Gly?Tyr?Cys?Pro?Thr?Met?Thr?Arg?Val?Leu?Gln?Gly?Val
35??????????????????40??????????????????45
Leu?Pro?Ala?Leu?Pro?Gln?Val?Val?Cys?Asn?Tyr?Arg?Asp?Val?Arg?Phe
50??????????????????55??????????????????60
Glu?Ser?Ile?Arg?Leu?Pro?Gly?Cys?Pro?Arg?Gly?Val?Asn?Pro?Val?Val
65??????????????????70??????????????????75??????????????????80
Ser?Tyr?Ala?Val?Ala?Leu?Ser?Cys?Gln?Cys?Ala?Leu?Cys?Arg?Arg?Ser
85??????????????????90??????????????????95
Thr?Thr?Asp?Cys?Gly?Gly?Pro?Lys?Asp?His?Pro?Leu?Thr?Cys?Asp?Asp
100?????????????????105?????????????????110
Pro?Arg?Phe?Gln?Asp?Ser?Ser?Ser?Ser?Lys?Ala?Pro?Pro?Pro?Ser?Leu
115?????????????????120?????????????????125
Pro?Ser?Pro?Ser?Arg?Leu?Pro?Gly?Pro?Cys?Asp?Thr?Pro?Ile?Leu?Pro
130?????????????????135?????????????????140
Gln?His?Pro?Glu?Thr?Leu?Val?Lys?Val?Lys?Asp?Ala?Glu?Asp?Gln?Leu
145?????????????????150?????????????????155?????????????????160
Gly?Ala?Arg?Val?Gly?Tyr?Ile?Glu?Leu?Asp?Leu?Asn?Ser?Gly?Lys?Ile
165?????????????????170?????????????????175
Leu?Glu?Ser?Phe?Arg?Pro?Glu?Glu?Arg?Phe?Pro?Met?Met?Ser?Thr?Phe
180?????????????????185?????????????????190
Lys?Val?Leu?Leu?Cys?Gly?Ala?Val?Leu?Ser?Arg?Ile?Asp?Ala?Gly?Gln
195?????????????????200?????????????????205
Glu?Gln?Leu?Gly?Arg?Arg?Ile?His?Tyr?Ser?Gln?Asn?Asp?Leu?Val?Glu
210?????????????????215?????????????????220
Tyr?Ser?Pro?Val?Thr?Glu?Lys?His?Leu?Thr?Asp?Gly?Met?Thr?Val?Arg
225?????????????????230?????????????????235?????????????????240
Glu?Leu?Cys?Ser?Ala?Ala?Ile?Thr?Met?Ser?Asp?Asn?Thr?Ala?Ala?Asn
245?????????????????250?????????????????255
Leu?Leu?Leu?Thr?Thr?Ile?Gly?Gly?Pro?Lys?Glu?Leu?Thr?Ala?Phe?Leu
260?????????????????265?????????????????270
His?Asn?Met?Gly?Asp?His?Val?Thr?Arg?Leu?Asp?Arg?Trp?Glu?Pro?Glu
275?????????????????280?????????????????285
Leu?Asn?Glu?Ala?Ile?Pro?Asn?Asp?Glu?Arg?Asp?Thr?Thr?Met?Pro?Val
290?????????????????295?????????????????300
Ala?Met?Ala?Thr?Thr?Leu?Arg?Lys?Leu?Leu?Thr?Gly?Glu?Leu?Leu?Thr
305?????????????????310?????????????????315?????????????????320
Leu?Ala?Ser?Arg?Gln?Gln?Leu?Ile?Asp?Trp?Met?Glu?Ala?Asp?Lys?Val
325?????????????????330?????????????????335
Ala?Gly?Pro?Leu?Leu?Arg?Ser?Ala?Leu?Pro?Ala?Gly?Trp?Phe?Ile?Ala
340?????????????????345?????????????????350
Asp?Lys?Ser?Gly?Ala?Gly?Glu?Arg?Gly?Ser?Arg?Gly?Ile?Ile?Ala?Ala
355?????????????????360?????????????????365
Lsu?Gly?Pro?Asp?Gly?Lys?Pro?Ser?Arg?Ile?Val?Val?Ile?Tyr?Thr?Thr
370?????????????????375?????????????????380
Gly?Ser?Gln?Ala?Thr?Met?Asp?Glu?Arg?Aen?Arg?Gln?Ile?Ala?Glu?Ile
385?????????????????390??????????????????395?????????????????400
Gly?Ala?Ser?Leu?Ile?Lys?His?Trp
405
<210>45
<211>125
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCGbeta，delta116-135，S138C
<400>45
Ser?Lys?Glu?Pro?Leu?Arg?Pro?Arg?Cys?Arg?Pro?Ile?Asn?Ala?Thr?Leu
1???????????????5???????????????????10??????????????????15
Ala?Val?Glu?Lys?Glu?Gly?Cys?Pro?Val?Cys?Ile?Thr?Val?Asn?Thr?Thr
20??????????????????25??????????????????30
Ile?Cys?Ala?Gly?Tyr?Cys?Pro?Thr?Met?Thr?Arg?Val?Leu?Gln?Gly?Val
35??????????????????40??????????????????45
Leu?Pro?Ala?Leu?Pro?Gln?Val?Val?Cys?Asn?Tyr?Arg?Asp?Val?Arg?Phe
50??????????????????55??????????????????60
Glu?Ser?Ile?Arg?Leu?Pro?Gly?Cys?Pro?Arg?Gly?Val?Asn?Pro?Val?Val
65??????????????????70??????????????????75??????????????????80
Ser?Tyr?Ala?Val?Ala?Leu?Ser?Cys?Gln?Cys?Ala?Leu?Cys?Arg?Arg?Ser
85??????????????????90??????????????????95
Thr?Thr?Asp?Cys?Gly?Gly?Pro?Lys?Asp?His?Pro?Leu?Thr?Cys?Asp?Asp
100?????????????????105?????????????????110
Pro?Arg?Phe?Gly?Pro?Cys?Asp?Thr?Pro?Ile?Leu?Pro?Gln
115?????????????????120
<210>46
<211>130
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCGbeta，delta121-135，S138C
<400>46
Ser?Lys?Glu?Pro?Leu?Arg?Pro?Arg?Cys?Arg?Pro?Ile?Asn?Ala?Thr?Leu
1???????????????5???????????????????10??????????????????15
Ala?Val?Glu?Lys?Glu?Gly?Cys?Pro?Val?Cys?Ile?Thr?Val?Asn?Thr?Thr
20??????????????????25??????????????????30
Ile?Cys?Ala?Gly?Tyr?Cys?Pro?Thr?Met?Thr?Arg?Val?Leu?Gln?Gly?Val
35??????????????????40??????????????????45
Leu?Pro?Ala?Leu?Pro?Gln?Val?Val?Cys?Asn?Tyr?Ars?Asp?Val?Arg?Phe
50??????????????????55??????????????????60
Glu?Ser?Ile?Arg?Leu?Pro?Gly?Cys?Pro?Arg?Gly?Val?Asn?Pro?Val?Val
65??????????????????70??????????????????75??????????????????80
Ser?Tyr?Ala?Val?Ala?Leu?Ser?Cys?Gln?Cys?Ala?Leu?Cys?Arg?Arg?Ser
85??????????????????90??????????????????95
Thr?Thr?Aep?Cys?Gly?Gly?Pro?Lys?Asp?His?Pro?Leu?Thr?Cys?Asp?Asp
100?????????????????105?????????????????110
Pro?Arg?Phe?Gln?Asp?Ser?Ser?Ser?Gly?Pro?Cys?Asp?Thr?Pro?Ile?Leu
115?????????????????120?????????????????125
Pro?Gln
<210>47
<211>136
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCGbeta，delta126-135，S138C
<400>47
Ser?Lys?Glu?Pro?Leu?Arg?Pro?Arg?Cys?Arg?Pro?Ile?Asn?Ala?Thr?Leu
1???????????????5???????????????????10??????????????????15
Ala?Val?Glu?Lys?Glu?Gly?Cys?Pro?Val?Cys?Ile?Thr?Val?Asn?Thr?Thr
20??????????????????25??????????????????30
Ile?Cys?Ala?Gly?Tyr?Cys?Pro?Thr?Met?Thr?Arg?Val?Leu?Gln?Gly?Val
35??????????????????40??????????????????45
Leu?Pro?Ala?Leu?Pro?Gln?Val?Val?Cys?Asn?Tyr?Arg?Asp?Val?Arg?Phe
50??????????????????55??????????????????60
Glu?Ser?Ile?Arg?Leu?Pro?Gly?Cys?Pro?Arg?Gly?Val?Asn?Pro?Val?Val
65??????????????????70??????????????????75??????????????????80
Ser?Tyr?Ala?Val?Ala?Leu?Ser?Cys?Gln?Cys?Ala?Leu?Cys?Arg?Arg?Ser
85??????????????????90??????????????????95
Thr?Thr?Asp?Cys?Gly?Gly?Pro?Lys?Asp?His?Pro?Leu?Thr?Cys?Asp?Asp
100?????????????????105?????????????????110
Pro?Arg?Phe?Gln?Asp?Ser?Ser?Ser?Ser?Lys?Ala?Pro?Pro?Pro?Gly?Pro
115?????????????????120?????????????????125
Cys?Asp?Thr?ProIle?Leu?Pro?Gln
130?????????????????135
<210>48
<211>140
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCGbeta，delta131-135，S138C
<400>48
Ser?Lys?Glu?Pro?Leu?Arg?Pro?Arg?Cys?Arg?Pro?Ile?Asn?Ala?Thr?Leu
1???????????????5???????????????????10??????????????????15
Ala?Val?Glu?Lys?Glu?Gly?Cys?Pro?Val?Cys?Ile?Thr?Val?Asn?Thr?Thr
20??????????????????25??????????????????30
Ile?Cys?Ala?Gly?Tyr?Cys?Pro?Thr?Met?Thr?Arg?Val?Leu?Gln?Gly?Val
35??????????????????40??????????????????45
Leu?Pro?Ala?Leu?Pro?Gln?Val?Val?Cys?Asn?Tyr?Arg?Asp?Val?Arg?Phe
50??????????????????55??????????????????60
Glu?Ser?Ile?Arg?Leu?Pro?Gly?Cys?Pro?Arg?Gly?Val?Asn?Pro?Val?Val
65??????????????????70??????????????????75??????????????????80
Ser?Tyr?Ala?Val?Ala?Leu?Ser?Cys?Gln?Cys?Ala?Leu?Cys?Arg?Arg?Ser
85??????????????????90??????????????????95
Thr?Thr?Asp?Cys?Gly?Gly?Pro?Lys?Asp?His?Pro?Leu?Thr?Cys?Asp?Asp
100?????????????????105?????????????????110
Pro?Arg?Phe?Gln?Asp?Ser?Ser?Ser?Ser?Lys?Ala?Pro?Pro?Pro?Ser?Leu
115?????????????????120?????????????????125
Pro?Ser?Gly?Pro?Cys?Asp?Thr?Pro?Ile?Leu?Pro?Gln
130?????????????????135
<210>49
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit，Lys91?replaced?with?Glu
<400>49
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Glu?Ser
85??????????????????90
<210>50
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?loop?2，Lys91?replaced?with?Met
<400>50
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Met?Ser
85??????????????????90
<210>51
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?loop?2，Lys44?replaced?with?Ala
<400>51
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Ala?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>52
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?loop?2，Lys44?replaced?with?Glu?and?Lys45?repla
ced?with?Gln
<400>52
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Glu?Gln?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>53
<211>92
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?alpha-subunit?loop?2，Lys44?replaced?with?Arg
<400>53
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Arg?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>54
<211>139
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?analog-beta101-145，alpha，residues?3-100?deleted?from?hCG
beta-subunit?with?alpha-subunit?fused?to?the?end?of?the?remaining
beta-subunit
<400>54
Ser?Lys?Gly?Gly?Pro?Lys?Asp?His?Pro?Leu?Thr?Cys?Asp?Asp?Pro?Arg
1???????????????5???????????????????10??????????????????15
Phe?Gln?Asp?Ser?Ser?Ser?Ser?Lys?Ala?Pro?Pro?Pro?Ser?Leu?Pro?Ser
20??????????????????25??????????????????30
Pro?Ser?Arg?Leu?Pro?Gly?Pro?Ser?Asp?Thr?Pro?Ile?Leu?Pro?Gln?Ala
35??????????????????40??????????????????45
Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro?Phe
50??????????????????55??????????????????60
Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys?Phe
65??????????????????70??????????????????75??????????????????80
Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu?Val
85??????????????????90??????????????????95
Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser?Tyr
100?????????????????105?????????????????110
Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr?Ala
115?????????????????120?????????????????125
Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
130?????????????????135
<210>55
<211>31
<212>PRT
<213>Homo?sapiens
<400>55
Phe?Gln?Asp?Ser?Ser?Ser?Ser?Lys?Ala?Pro?Pro?Pro?Ser?Leu?Pro?Ser
1???????????????5???????????????????10??????????????????15
Pro?Ser?Arg?Leu?Pro?Gly?Pro?Ser?Thr?Asp?Pro?Ile?Leu?Pro?Gly
20??????????????????25??????????????????30
<210>56
<211>10
<212>PRT
<213>Artificial?Sequence
<220>
<223>Xl-Asp-Asp-Asp-Asp-Lys-Ser-Ym-Cys-Zn，where?X，Y，and?Z?refer?to
any?tail?portion?amino?acids?and?l，m，and?n?refer?to?the?lengths
of?the?tail?portion?amino?acids
<220>
<221>MISC_FEATURE
<223>xaa?refers?to?any?tail?portion?amino?acids?and?n?refers?to?the
lengths?of?the?tail?portion?amino?acids
<400>56
xaa
n?Asp?Asp?Asp?Asp?Lys?Ser?xaa
n?Cys?xaa
n
1???????????????????5?????????????????????10
<210>57
<211>92
<212>PRT
<213>Artifical?Sequence
<220>
<223>An?hCG?truncated?β-aubunit?analog?fused?to?the?hCG?alpha-carboxyterminus
<400>57
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser?Asp?Asp?Pro?Arg
85??????????????????90??????????????????95
Phe?Gly?Pro?Cys?Asp?Thr?Pro?Ile?Leu?Pro?Gln
100?????????????????105
<210>58
<211>145
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?beta-subunit?with?Cys?substituted?for?Arg94
<400>58
Ser?Lys?Glu?Pro?Leu?Arg?Pro?Arg?Cys?Arg?Pro?Ile?Asn?Ala?Thr?Leu
1???????????????5???????????????????10??????????????????15
Ala?Val?Glu?Lys?Glu?Gly?Cys?Pro?Val?Cys?Ile?Thr?Val?Asn?Thr?Thr
20??????????????????25??????????????????30
Ile?Cys?Ala?Gly?Tyr?Cys?Pro?Thr?Met?Thr?Arg?Val?Leu?Gln?Gly?Val
35??????????????????40??????????????????45
Leu?Pro?Ala?Leu?Pro?Gln?Val?Val?Cys?Asn?Tyr?Arg?Asp?Val?Arg?Phe
50??????????????????55??????????????????60
Glu?Ser?Ile?Arg?Leu?Pro?Gly?Cys?Pro?Arg?Gly?Val?Pro?Asn?Val?Val
65??????????????????70??????????????????75???????????????????80
Ser?Tyr?Ala?Val?Ala?Leu?ser?Cys?Gln?Cys?Ala?Leu?Cys?Cys?Arg?Ser
85??????????????????90??????????????????95
Thr?Tbr?Asp?Cys?Gly?Gly?Pro?Lys?Asp?His?Pro?Leu?Thr?Cys?Asp?Asp
100?????????????????105?????????????????110
Pro?Arg?Phe?Gln?Asp?Ser?Ser?Ser?Ser?Lys?Ala?Pro?Pro?Pro?Ser?Leu
115?????????????????120?????????????????125
Pro?Ser?Pro?Ser?Arg?Leu?Pro?Gly?Pro?Ser?Asp?Thr?Pro?Ile?Leu?Pro?Gln
130?????????????????135?????????????????140
<210>59
<211>145
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?beta-subunit?with?Cys?substituted?for?Arg95
<400>59
Ser?Lys?Glu?Pro?Leu?Arg?Pro?Arg?Cys?Arg?Pro?Ile?Asn?Ala?Thr?Leu
1???????????????5???????????????????10??????????????????15
Ala?Val?Glu?Lys?Glu?Gly?Cys?Pro?Val?Cys?Ile?Thr?Val?Asn?Thr?Thr
20??????????????????25??????????????????30
Ile?Cys?Ala?Gly?Tyr?Cys?Pro?Thr?Met?Thr?Arg?Val?Leu?Gln?Gly?Val
35??????????????????40??????????????????45
Leu?Pro?Ala?Leu?Pro?Gln?Val?Val?Cys?Asn?Tyr?Arg?Asp?Val?Arg?Phe
50??????????????????55??????????????????60
Glu?Ser?Ile?Arg?Leu?Pro?Gly?Cys?Pro?Arg?Gly?Val?Pro?Asn?Val?Val
65??????????????????70??????????????????75???????????????????80
Ser?Tyr?Ala?Val?Ala?Leu?Ser?Cys?Gln?Cys?Ala?Leu?Cys?Arg?Cys?Ser
85??????????????????90??????????????????95
Thr?Thr?Asp?Cys?Gly?Gly?Pro?Lys?Asp?His?Pro?Leu?Thr?Cys?Asp?Asp
100?????????????????105?????????????????110
Pro?Arg?Phe?Gln?Asp?Ser?Ser?Ser?Ser?Lys?Ala?Pro?Pro?Pro?Ser?Leu
115?????????????????120?????????????????125
Pro?Ser?Pro?Ser?Arg?Leu?Pro?Gly?Pro?Ser?Asp?Thr?Pro?Ile?Leu?Pro?Gln
130?????????????????135?????????????????140
<210>60
<211>145
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?beta-subunit?with?Cys?substituted?for?Ser96
<400>60
Ser?Lys?Glu?Pro?Leu?Arg?Pro?Arg?Cys?Arg?Pro?Ile?Asn?Ala?Thr?Leu
1???????????????5???????????????????10??????????????????15
Ala?Val?Glu?Lys?Glu?Gly?Cys?Pro?Val?Cys?Ile?Thr?Val?Asn?Thr?Thr
20??????????????????25??????????????????30
Ile?Cys?Ala?Gly?Tyr?Cys?Pro?Thr?Met?Thr?Arg?Val?Leu?Gln?Gly?Val
35??????????????????40??????????????????45
Leu?Pro?Ala?Leu?Pro?Gln?Val?Val?Cys?Asn?Tyr?Arg?Asp?Val?Arg?Phe
50??????????????????55??????????????????60
Glu?Ser?Ile?Arg?Leu?Pro?Gly?Cys?Pro?Arg?Gly?Val?Pro?Asn?Val?Val
65??????????????????70??????????????????75??????????????????80
Ser?Tyr?Ala?Val?Ala?Leu?Ser?Cys?Gln?Cys?Ala?Leu?Cys?Arg?Arg?Cys
85??????????????????90??????????????????95
Thr?Thr?Asp?Cys?Gly?Gly?Pro?Lys?Asp?His?Pro?Leu?Thr?Cys?Asp?Asp
100?????????????????105?????????????????110
Pro?Arg?Phe?Gln?Asp?Ser?Ser?Ser?Ser?Lys?Ala?Pro?Pro?Pro?Ser?Leu
115?????????????????120?????????????????125
Pro?Ser?Pro?Ser?Arg?Leu?Pro?Gly?Pro?Ser?Asp?Thr?Pro?Ile?Leu?Pro?Gln
130?????????????????135?????????????????140
<210>61
<211>145
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?beta-subunit?with?Cys?substituted?for?Thr97
<400>61
Ser?Lys?Glu?Pro?Leu?Arg?Pro?Arg?Cys?Arg?Pro?Ile?Asn?Ala?Thr?Leu
1???????????????5???????????????????10??????????????????15
Ala?Val?Glu?Lys?Glu?Gly?Cys?Pro?Val?Cys?Ile?Thr?Val?Asn?Thr?Thr
20??????????????????25??????????????????30
Ile?Cys?Ala?Gly?Tyr?Cys?Pro?Thr?Met?Thr?Arg?Val?Leu?Gln?Gly?Val
35??????????????????40??????????????????45
Leu?Pro?Ala?Leu?Pro?Gln?Val?Val?Cys?Asn?Tyr?Arg?Asp?Val?Arg?Phe
50??????????????????55??????????????????60
Glu?Ser?Ile?Arg?Leu?Pro?Gly?Cys?Pro?Arg?Gly?Val?Pro?Asn?Val?Val
65??????????????????70??????????????????75??????????????????80
Ser?Tyr?Ala?Val?Ala?Leu?Ser?Cys?Gln?Cys?Ala?Leu?Cys?Arg?Arg?Ser
85??????????????????90??????????????????95
Cys?Thr?Asp?Cys?Gly?Gly?Pro?Lys?Asp?His?Pro?Leu?Thr?Cys?Asp?Asp
100?????????????????105?????????????????110
Pro?Arg?Phe?Gln?Asp?Ser?Ser?Ser?Ser?Lys?Ala?Pro?Pro?Pro?Ser?Leu
115????????????????120??????????????????125
Pro?Ser?Pro?Ser?Arg?Leu?Pro?Gly?Pro?Ser?Asp?Thr?Pro?Ile?Leu?Pro?Gln
130?????????????????135?????????????????140
<210>62
<211>145
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?beta-subunit?with?Cys?substituted?for?Thr98
<400>62
Ser?Lys?Glu?Pro?Leu?Arg?Pro?Arg?Cys?Arg?Pro?Ile?Asn?Ala?Thr?Leu
1???????????????5???????????????????10??????????????????15
Ala?Val?Glu?Lys?Glu?Gly?Cys?Pro?Val?Cys?Ile?Thr?Val?Asn?Thr?Thr
20??????????????????25??????????????????30
Ile?Cys?Ala?Gly?Tyr?Cys?Pro?Thr?Met?Thr?Arg?Val?Leu?Gln?Gly?Val
35??????????????????40??????????????????45
Leu?Pro?Ala?Leu?Pro?Gln?Val?Val?Cys?Asn?Tyr?Arg?Asp?Val?Arg?Phe
50??????????????????55??????????????????60
Glu?Ser?Ile?Arg?Leu?Pro?Gly?Cys?Pro?Arg?Gly?Val?Pro?Asn?Val?Val
65??????????????????70??????????????????75??????????????????80
Ser?Tyr?Ala?Val?Ala?Leu?Ser?Cys?Gln?Cys?Ala?Leu?Cys?Arg?Arg?Ser
85??????????????????90??????????????????95
Thr?Cys?Asp?Cys?Gly?Gly?Pro?Lys?Asp?His?Pro?Leu?Thr?Cys?Asp?Asp
100?????????????????105?????????????????110
Pro?Arg?Phe?Gln?Asp?Ser?Ser?Ser?Ser?Lys?Ala?Pro?Pro?Pro?Ser?Leu
115?????????????????120?????????????????125
Pro?Ser?Pro?Ser?Arg?Leu?Pro?Gly?Pro?Ser?Asp?Thr?Pro?Ile?Leu?Pro?Gln
130?????????????????135?????????????????140
<210>63
<211>145
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?beta-subunit?with?Cys?substituted?for?Asp99
<400>63
Ser?Lys?Glu?Pro?Leu?Arg?Pro?Arg?Cys?Arg?Pro?Ile?Asn?Ala?Thr?Leu
1???????????????5???????????????????10??????????????????15
Ala?Val?Glu?Lys?Glu?Gly?Cys?Pro?Val?Cys?Ile?Thr?Val?Asn?Thr?Thr
20??????????????????25??????????????????30
Ile?Cys?Ala?Gly?Tyr?Cys?Pro?Thr?Met?Thr?Arg?Val?Leu?Gln?Gly?Val
35??????????????????40??????????????????45
Leu?Pro?Ala?Leu?Pro?Gln?Val?Val?Cys?Asn?Tyr?Arg?Asp?Val?Arg?Phe
50??????????????????55??????????????????60
Glu?Ser?Ile?Arg?Leu?Pro?Gly?Cys?Pro?Arg?Gly?Val?Pro?Asn?Val?Val
65??????????????????70??????????????????75??????????????????80
Ser?Tyr?Ala?Val?Ala?Leu?Ser?Cys?Gln?Cys?Ala?Leu?Cys?Arg?Arg?Ser
85??????????????????90??????????????????95
Thr?Thr?Cys?Cys?Gly?Gly?Pro?Lys?Asp?His?Pro?Leu?Thr?Cys?Asp?Asp
100?????????????????105?????????????????110
Pro?Arg?Phe?Gln?Asp?Ser?Ser?Ser?Ser?Lys?Ala?Pro?Pro?Pro?Ser?Leu
115?????????????????120?????????????????125
Pro?Ser?Pro?Ser?Arg?Leu?Pro?Gly?Pro?Ser?Asp?Thr?Pro?Ile?Leu?Pro?Gln
130?????????????????135?????????????????140
<210>64
<211>95
<212>PRT
<213>Artifical?Sequence
<220>
<223>An?hCG?alpha-subunit?analog?with?Gly-Gly-cys?at?its?carboxyterminus
<400>64
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asn?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser?Gly?Gly?Cys
86??????????????????90??????????????????95
<210>65
<211>92
<212>PRT
<213>Artifical?Sequence
<220>
<223>An?hCG?alpha-subunit?analog?with?Asp?in?place?of?Asn52?and?Cys?in?place?of
Ser92
<400>65
Ala?Pro?Asp?Val?Gln?Asp?Cys?Pro?Glu?Cys?Thr?Leu?Gln?Glu?Asn?Pro
1???????????????5???????????????????10??????????????????15
Phe?Phe?Ser?Gln?Pro?Gly?Ala?Pro?Ile?Leu?Gln?Cys?Met?Gly?Cys?Cys
20??????????????????25??????????????????30
Phe?Ser?Arg?Ala?Tyr?Pro?Thr?Pro?Leu?Arg?Ser?Lys?Lys?Thr?Met?Leu
35??????????????????40??????????????????45
Val?Gln?Lys?Asp?Val?Thr?Ser?Glu?Ser?Thr?Cys?Cys?Val?Ala?Lys?Ser
50??????????????????55??????????????????60
Tyr?Asn?Arg?Val?Thr?Val?Met?Gly?Gly?Phe?Lys?Val?Glu?Asn?His?Thr
65??????????????????70??????????????????75??????????????????80
Ala?Cys?His?Cys?Ser?Thr?Cys?Tyr?Tyr?His?Lys?Ser
85??????????????????90
<210>66
<211>145
<212>PRT
<213>Artificial?Sequence
<220>
<223>hCG?beta-subunit?with?Cys?substituted?for?Ser96?and?hFSH?beta-subunit
residues?95-108?for?hCG?beta-subunit?residues?101-108
<400>66
Ser?Lys?Glu?Pro?Leu?Arg?Pro?Arg?Cys?Arg?Pro?Ile?Asn?Ala?Thr?Leu
1???????????????5???????????????????10??????????????????15
Ala?Val?Glu?Lys?Glu?Gly?Cys?Pro?Val?Cys?Ile?Thr?Val?Asn?Thr?Thr
20??????????????????25??????????????????30
Ile?Cys?Ala?Gly?Tyr?Cys?Pro?Thr?Met?Thr?Arg?Val?Leu?Gln?Gly?Val
35??????????????????40??????????????????45
Leu?Pro?Ala?Leu?Pro?Gln?Val?Val?Cys?Asn?Tyr?Arg?Asp?Val?Arg?Phe
50??????????????????55??????????????????60
Glu?Ser?Ile?Arg?Leu?Pro?Gly?Cys?Pro?Arg?Gly?Val?Pro?Asn?Val?Val
65??????????????????70??????????????????75??????????????????80
Ser?Tyr?Ala?Val?Ala?Leu?Ser?Cys?Gln?Cys?Ala?Leu?Cys?Arg?Arg?Cys
85??????????????????90??????????????????95
Thr?Thr?Asp?Cys?Thr?Val?Arg?Gly?Leu?Gly?Pro?Ser?Tyr?Cys?Ser?Phe
100?????????????????105?????????????????110
Gly?Glu?Phe?Gln?Asp?Ser?Ser?Ser?Ser?Lys?Ala?Pro?Pro?Pro?Ser?Leu
115?????????????????120?????????????????125
Pro?Ser?Pro?Ser?Arg?Leu?Pro?Gly?Pro?Ser?Asp?Thr?Pro?Ile?Leu?Pro?Gln
130??????????????????135?????????????????140
Claims (19)
1. composition comprises:
Protein portion, wherein this protein portion contains the alternate cysteine residues in the need mark;
Be positioned at the afterbody of protein portion end; And
Tubercle, wherein this tubercle is positioned at the free-end of afterbody, and contains cysteine residues, and nodular cysteine residues can form disulfide linkage with the alternate halfcystine of protein portion.
2. the composition of claim 1, wherein afterbody contains proteolytic enzyme cutting site.
3. the composition of claim 1, wherein tubercle comprises phenotypic markers.
4. the composition of claim 1, wherein tubercle comprises polypeptide.
5. the composition of claim 1, wherein tubercle comprises protein.
6. the composition of claim 5, wherein nodular halfcystine is positioned at proteinic surface.
7. the composition of claim 1, wherein protein portion is a monomeric protein.
8. the composition of claim 1, wherein protein portion is a polymer protein.
9. method at the specific site labelled protein comprises:
A) select desired protein;
B) want the mark part to position to desired protein;
C) select required tubercle, wherein required tubercle contains cysteine residues;
D) preparation coding desired protein, afterbody and required nodular construct, wherein desired protein is wanting the mark part to have alternate half Guang amino acid;
E) construct is imported in a kind of cell protein with presentation markup, wherein halfcystine in the tubercle and the alternate halfcystine in the desired protein form disulfide linkage.
10. the method in the claim 9, wherein tubercle comprises phenotypic markers, signal sequence or protein.
11. the method in the claim 10, wherein tubercle protein is proteolytic enzyme.
12. the method in the claim 9, wherein desired protein exists with monomeric form.
13. the method in the claim 9, wherein desired protein exists with the polymer form.
14. a method for purifying proteins comprises:
A) with effable construct transfered cell, this construct a kind of protein of encoding wherein, this protein is wanting the mark part to comprise the alternate halfcystine, comprise afterbody at proteinic end, this afterbody contains the cleavage site of proteolytic enzyme, at the terminal nodosity of afterbody, this tubercle contains cysteine residues;
B) lysing cell;
C) according to nodular feature protein purification.
15. the method for a mark hCG comprises:
A) preparation can be expressed the construct of natural hCG β or hCG β-S138C;
B) preparation can be expressed the construct of natural hCG α or the alternative analogue of hCG α halfcystine;
C) with step a) and b) construct of preparation imports in the COS-7 cell and carries out coexpression.
16. the method for claim 15, wherein the construct of step a) also comprises protein is merged mutually with the 140th or 145 residue of hCG β.
17. the method for claim 16, wherein protein is β-Nei Xiananmei.
18. the method for claim 15, wherein hCG α halfcystine substitutes analogue and is selected from SEQ ID NO:1 to SEQ ID NO:35.
19. a method of surveying and drawing distance between protein molecule comprises:
A) select first protein molecule;
B) select second protein molecule, wherein first protein molecule and second proteins interaction;
C) generate first protein molecule, every kind first protein molecule that is wherein produced contains a tubercle on the first proteinic different loci;
D) generate second protein molecule;
E) protein analysis first protein that utilizes step c) and step d) and generated and the distance between second protein.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US34528301P | 2001-11-08 | 2001-11-08 | |
| US60/345,283 | 2001-11-08 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1596304A true CN1596304A (en) | 2005-03-16 |
Family
ID=23354368
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA02822101XA Pending CN1596304A (en) | 2001-11-08 | 2002-11-08 | Protein knobs |
Country Status (13)
| Country | Link |
|---|---|
| EP (1) | EP1497634A4 (en) |
| JP (2) | JP2005509652A (en) |
| KR (1) | KR20040063928A (en) |
| CN (1) | CN1596304A (en) |
| CA (1) | CA2466595A1 (en) |
| EA (1) | EA200400647A1 (en) |
| HR (1) | HRP20040242A2 (en) |
| IL (1) | IL161804A0 (en) |
| MX (1) | MXPA04004289A (en) |
| NO (1) | NO20042358L (en) |
| PL (1) | PL373566A1 (en) |
| WO (1) | WO2003040695A2 (en) |
| ZA (1) | ZA200404373B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5014988B2 (en) * | 2004-07-16 | 2012-08-29 | マイクロメット アクツィエン ゲゼルシャフト | Expression enhancing polypeptide |
| GB201403815D0 (en) * | 2014-03-04 | 2014-04-16 | Mologic Ltd | Assay |
| CN112986571B (en) * | 2019-12-12 | 2023-04-11 | 中国科学院大连化学物理研究所 | Method for identifying Midkine spatiotemporal network interaction protein |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0578328B1 (en) * | 1983-11-02 | 1999-12-29 | Applied Research Systems ARS Holding N.V. | Material and methods for the production of a heterodimeric human LH with a Met42 Val55 alpha subunit |
| JP3045539B2 (en) * | 1989-02-21 | 2000-05-29 | ワシントン ユニバーシティ | Modified reproductive hormone |
| US5166322A (en) * | 1989-04-21 | 1992-11-24 | Genetics Institute | Cysteine added variants of interleukin-3 and chemical modifications thereof |
| WO1997004080A1 (en) * | 1995-07-14 | 1997-02-06 | Fuji Yakuhin Kogyo Kabushiki Kaisha | Novel protein and monoclonal antibody specific thereto |
| US6001354A (en) * | 1995-11-17 | 1999-12-14 | Washington University | GRB2 associating polypeptides and nucleic acids encoding therefor |
| US5837837A (en) * | 1997-02-27 | 1998-11-17 | Millennium Pharmaceuticals, Inc. | Nucleic acids molecules encoding Caspase-8h and Caspase-8i |
| DK1775346T3 (en) * | 1997-06-25 | 2009-09-28 | Merck Serono Sa | Disulfide crosslinked glycoprotein hormone analogues, their preparation and use |
-
2002
- 2002-11-08 IL IL16180402A patent/IL161804A0/en unknown
- 2002-11-08 EA EA200400647A patent/EA200400647A1/en unknown
- 2002-11-08 CN CNA02822101XA patent/CN1596304A/en active Pending
- 2002-11-08 MX MXPA04004289A patent/MXPA04004289A/en not_active Application Discontinuation
- 2002-11-08 EP EP02789525A patent/EP1497634A4/en not_active Withdrawn
- 2002-11-08 CA CA002466595A patent/CA2466595A1/en not_active Abandoned
- 2002-11-08 PL PL02373566A patent/PL373566A1/en not_active Application Discontinuation
- 2002-11-08 JP JP2003542901A patent/JP2005509652A/en active Pending
- 2002-11-08 WO PCT/US2002/035914 patent/WO2003040695A2/en active Application Filing
- 2002-11-08 KR KR10-2004-7006994A patent/KR20040063928A/en not_active Application Discontinuation
-
2004
- 2004-03-12 HR HR20040242A patent/HRP20040242A2/en not_active Application Discontinuation
- 2004-06-03 ZA ZA2004/04373A patent/ZA200404373B/en unknown
- 2004-06-07 NO NO20042358A patent/NO20042358L/en not_active Application Discontinuation
-
2007
- 2007-10-11 JP JP2007265961A patent/JP2008110973A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| ZA200404373B (en) | 2005-11-30 |
| HRP20040242A2 (en) | 2005-02-28 |
| WO2003040695A2 (en) | 2003-05-15 |
| PL373566A1 (en) | 2005-09-05 |
| IL161804A0 (en) | 2005-11-20 |
| EP1497634A2 (en) | 2005-01-19 |
| KR20040063928A (en) | 2004-07-14 |
| CA2466595A1 (en) | 2003-05-15 |
| NO20042358L (en) | 2004-08-09 |
| JP2005509652A (en) | 2005-04-14 |
| WO2003040695A3 (en) | 2004-11-11 |
| EP1497634A4 (en) | 2007-02-07 |
| JP2008110973A (en) | 2008-05-15 |
| MXPA04004289A (en) | 2005-03-31 |
| EA200400647A1 (en) | 2004-10-28 |
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