CN116804041A - Polypeptide for detecting residual quantity and activity of various proteases and application thereof - Google Patents
Polypeptide for detecting residual quantity and activity of various proteases and application thereof Download PDFInfo
- Publication number
- CN116804041A CN116804041A CN202310562097.4A CN202310562097A CN116804041A CN 116804041 A CN116804041 A CN 116804041A CN 202310562097 A CN202310562097 A CN 202310562097A CN 116804041 A CN116804041 A CN 116804041A
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- amino acid
- detecting
- detected
- proteases
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 108
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 83
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 76
- 108091005804 Peptidases Proteins 0.000 title claims abstract description 68
- 239000004365 Protease Substances 0.000 title claims abstract description 68
- 102000035195 Peptidases Human genes 0.000 title claims abstract description 67
- 230000000694 effects Effects 0.000 title claims abstract description 43
- 102000004190 Enzymes Human genes 0.000 claims abstract description 58
- 108090000790 Enzymes Proteins 0.000 claims abstract description 58
- 238000001514 detection method Methods 0.000 claims abstract description 46
- 150000001413 amino acids Chemical class 0.000 claims abstract description 35
- 238000000034 method Methods 0.000 claims abstract description 31
- 102000005593 Endopeptidases Human genes 0.000 claims abstract description 19
- 108010059378 Endopeptidases Proteins 0.000 claims abstract description 19
- 238000005520 cutting process Methods 0.000 claims abstract description 15
- -1 aromatic amino acid Chemical class 0.000 claims abstract description 14
- 102000018389 Exopeptidases Human genes 0.000 claims abstract description 7
- 108010091443 Exopeptidases Proteins 0.000 claims abstract description 7
- 150000001412 amines Chemical class 0.000 claims abstract description 6
- 229940088598 enzyme Drugs 0.000 claims description 53
- 235000001014 amino acid Nutrition 0.000 claims description 34
- 238000003776 cleavage reaction Methods 0.000 claims description 29
- 230000007017 scission Effects 0.000 claims description 29
- 239000000758 substrate Substances 0.000 claims description 26
- 238000006243 chemical reaction Methods 0.000 claims description 23
- 239000000523 sample Substances 0.000 claims description 21
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims description 16
- 108090000087 Carboxypeptidase B Proteins 0.000 claims description 15
- 102000003670 Carboxypeptidase B Human genes 0.000 claims description 15
- 108090000631 Trypsin Proteins 0.000 claims description 14
- 102000004142 Trypsin Human genes 0.000 claims description 14
- 238000001976 enzyme digestion Methods 0.000 claims description 14
- 239000012588 trypsin Substances 0.000 claims description 14
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 13
- 108010053229 Lysyl endopeptidase Proteins 0.000 claims description 11
- 239000000872 buffer Substances 0.000 claims description 10
- 239000012295 chemical reaction liquid Substances 0.000 claims description 10
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 10
- 239000013558 reference substance Substances 0.000 claims description 10
- 102000004877 Insulin Human genes 0.000 claims description 8
- 108090001061 Insulin Proteins 0.000 claims description 8
- 229940125396 insulin Drugs 0.000 claims description 8
- 239000004472 Lysine Substances 0.000 claims description 7
- 108010013369 Enteropeptidase Proteins 0.000 claims description 6
- 102100029727 Enteropeptidase Human genes 0.000 claims description 6
- PMZXXNPJQYDFJX-UHFFFAOYSA-N acetonitrile;2,2,2-trifluoroacetic acid Chemical compound CC#N.OC(=O)C(F)(F)F PMZXXNPJQYDFJX-UHFFFAOYSA-N 0.000 claims description 6
- 239000012488 sample solution Substances 0.000 claims description 6
- 239000004475 Arginine Substances 0.000 claims description 5
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 5
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 5
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 claims description 5
- 239000003814 drug Substances 0.000 claims description 5
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 4
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 4
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims description 4
- 102000004400 Aminopeptidases Human genes 0.000 claims description 3
- 108090000915 Aminopeptidases Proteins 0.000 claims description 3
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 claims description 3
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 claims description 3
- 102000005367 Carboxypeptidases Human genes 0.000 claims description 3
- 108010006303 Carboxypeptidases Proteins 0.000 claims description 3
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 3
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 3
- 235000012538 ammonium bicarbonate Nutrition 0.000 claims description 3
- 239000001099 ammonium carbonate Substances 0.000 claims description 3
- 235000003704 aspartic acid Nutrition 0.000 claims description 3
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 3
- 229940079593 drug Drugs 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 3
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 3
- 108090000317 Chymotrypsin Proteins 0.000 claims description 2
- 229940089838 Glucagon-like peptide 1 receptor agonist Drugs 0.000 claims description 2
- 101800000221 Glucagon-like peptide 2 Proteins 0.000 claims description 2
- 102000057297 Pepsin A Human genes 0.000 claims description 2
- 108090000284 Pepsin A Proteins 0.000 claims description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 2
- 229960002376 chymotrypsin Drugs 0.000 claims description 2
- 239000000945 filler Substances 0.000 claims description 2
- 239000003877 glucagon like peptide 1 receptor agonist Substances 0.000 claims description 2
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical group CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 2
- 229940111202 pepsin Drugs 0.000 claims description 2
- 239000013641 positive control Substances 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 239000000741 silica gel Substances 0.000 claims description 2
- 229910002027 silica gel Inorganic materials 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 39
- 239000007788 liquid Substances 0.000 description 17
- 239000000047 product Substances 0.000 description 15
- 239000000543 intermediate Substances 0.000 description 12
- YSDQQAXHVYUZIW-QCIJIYAXSA-N Liraglutide Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCNC(=O)CC[C@H](NC(=O)CCCCCCCCCCCCCCC)C(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=C(O)C=C1 YSDQQAXHVYUZIW-QCIJIYAXSA-N 0.000 description 11
- 108010019598 Liraglutide Proteins 0.000 description 11
- 229960002701 liraglutide Drugs 0.000 description 11
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 8
- 230000008569 process Effects 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 5
- 238000004364 calculation method Methods 0.000 description 5
- 241000700124 Octodon degus Species 0.000 description 3
- 102000007079 Peptide Fragments Human genes 0.000 description 3
- 108010033276 Peptide Fragments Proteins 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 102000000496 Carboxypeptidases A Human genes 0.000 description 2
- 108010080937 Carboxypeptidases A Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 101710172072 Kexin Proteins 0.000 description 2
- 101001018085 Lysobacter enzymogenes Lysyl endopeptidase Proteins 0.000 description 2
- PWUBONDMIMDOQY-UHFFFAOYSA-N acetonitrile;hydrochloride Chemical compound Cl.CC#N PWUBONDMIMDOQY-UHFFFAOYSA-N 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 125000000623 heterocyclic group Chemical group 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 108091005508 Acid proteases Proteins 0.000 description 1
- 102000035101 Aspartic proteases Human genes 0.000 description 1
- 108091005502 Aspartic proteases Proteins 0.000 description 1
- 108090000145 Bacillolysin Proteins 0.000 description 1
- 108091005658 Basic proteases Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102100031780 Endonuclease Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000005741 Metalloproteases Human genes 0.000 description 1
- 108010006035 Metalloproteases Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000035092 Neutral proteases Human genes 0.000 description 1
- 108091005507 Neutral proteases Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 102000029797 Prion Human genes 0.000 description 1
- 108091000054 Prion Proteins 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 101710097834 Thiol protease Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 150000001371 alpha-amino acids Chemical class 0.000 description 1
- 235000008206 alpha-amino acids Nutrition 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000009748 deglutition Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- SDIXRDNYIMOKSG-UHFFFAOYSA-L disodium methyl arsenate Chemical compound [Na+].[Na+].C[As]([O-])([O-])=O SDIXRDNYIMOKSG-UHFFFAOYSA-L 0.000 description 1
- 238000007905 drug manufacturing Methods 0.000 description 1
- 229940066758 endopeptidases Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
Abstract
The invention provides a polypeptide for detecting the residual quantity and activity of various proteases, which comprises at least one enzyme cutting site, wherein the number of amino acids contained in the polypeptide is a,3< a <20; at least one of the amino acids is one of aromatic amino acid and derivatives thereof, heterocyclic amino acid and derivatives thereof and is positioned at the N end or the C end of the enzyme cutting site; the enzyme cutting sites comprise one or more of endopeptidase enzyme cutting sites and exopeptidase enzyme cutting sites. The method has high sensitivity, the detection limit of the polypeptide is 0.03125mmol/L, and can detect various proteases at one time, and the detection limit of the protease can reach mug level or below, thereby being beneficial to detecting the enzyme activity under the conditions of lower enzyme activity and fewer products.
Description
Technical Field
The invention relates to the technical field of chromatographic detection, in particular to a polypeptide for detecting the residual quantity and activity of various proteases and application thereof.
Background
Peptides (peptides) are compounds formed by peptide bonds of alpha-amino acids, which are also proteolytic intermediates. Polypeptides are biologically active substances that are involved in various cellular functions in an organism. With the rapid development of molecular biology and biochemical technology, the research of polypeptides has been advanced in a striking and epoch-making way. It has been found that there are tens of thousands of polypeptides present in organisms, and that all cells are able to synthesize the polypeptides. At present, the application of the polypeptide is mainly focused on polypeptide medicaments, polypeptide medicament carriers, polypeptide nutritional foods and the like.
Proteases are a generic term for a class of enzymes that hydrolyze protein peptide chains. They are classified into endopeptidases and exopeptidases according to the manner in which they degrade the polypeptide. The former can cleave polypeptide chain with large molecular weight from the middle to form prion with smaller molecular weight and peptone; the latter can be further divided into carboxypeptidase and aminopeptidase which hydrolyze the peptide chain one by one from the free carboxy-terminus or the free amino-terminus of the polypeptide, respectively, to form amino acids. Proteases can be further classified by nature into serine proteases, thiol proteases, metalloproteases and aspartic proteases. According to the optimal pH value of the reaction, the method is divided into acid protease, neutral protease and alkaline protease. Proteases are widely found in animals, plants and microorganisms. The natural protease can be obtained by direct extraction, and the recombinant protease can also be obtained by genetic engineering technology. Proteases have been widely used in industry, agriculture, food, and in particular in the pharmaceutical industry.
Currently, in the process of recombinant protein drug manufacturing, such as insulin and analogues thereof, carboxypeptidase B (CPB), double-base amino acid endopeptidase (Kex 2, kexin), lysyl endopeptidase (Lys-C), trypsin (Trypsin) and the like are adopted according to different processes and designs; in the process of the recombinant polypeptide, carboxypeptidase B (CPB), carboxypeptidase A (CPA), double-base amino acid endopeptidase (Kex 2, kexin), lysyl endopeptidase (Lys-C/Lys-N), trypsin (Trypsin) and the like are respectively adopted according to different processes and designs. The current use of these proteases in pharmaceutical processes is to release the peptide chain or to specifically cleave the C/N-terminus of the peptide chain.
The protease is directly added in the process, and then the inactivation or removal process is carried out. There may be a trace amount of protease residue, so that the protease residue needs to be detected for the product. At present, an enzyme-linked immunosorbent assay (ELISA) method can achieve a certain residual quantity detection, but cannot detect the enzyme activity residual quantity; the method has the problems of weak specificity, possibility of interference by products and the like.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides a polypeptide for detecting the residual quantity and the activity of various proteases and application thereof, which solves the problem that the residual quantity of the enzyme activity cannot be detected in the prior art; the method has the problems of weak specificity and possibility of being interfered by products.
In one aspect of the invention, a polypeptide for detecting the residual amount and activity of various proteases is provided, wherein the polypeptide comprises at least one enzyme cutting site, and the number of amino acids contained in the polypeptide is a,3< a <20; at least one of the amino acids is one of aromatic amino acid and derivatives thereof, heterocyclic amino acid and derivatives thereof and is positioned at the N end or the C end of the enzyme cutting site; the enzyme cutting sites comprise one or more of endopeptidase cutting sites and exopeptidase cutting sites;
preferably, the aromatic amino acids include tyrosine, tryptophan, phenylalanine; the heterocyclic amino acid includes histidine; the endopeptidase comprises lysyl endopeptidase, trypsin, pepsin, enterokinase, double-base amino acid endopeptidase, chymotrypsin; the exopeptidase includes carboxypeptidase and aminopeptidase.
The cleavage sites for proteases are shown in the following table:
note that: in the table, K represents lysine, R represents arginine, D represents aspartic acid, W represents tryptophan, F represents phenylalanine, and P represents proline
Further, the amino acid sequence of the polypeptide is X 1 -X 2 -K 1 -R-X 3 -X 4 -K 2 ，
X 1 、X 2 、X 3 、X 4 Is amino acid, K 1 、K 2 Each is lysine and R is arginine;
wherein X is 2 -K 1 K is a cleavage site of lysyl endopeptidase and trypsin 1 R is a double-base amino acid endopeptidase, trypsin cleavage site, X 4 -K 2 Is carboxypeptidase B cleavage site;
preferably, the amino acid sequence of the polypeptide is YGKRLWK. As shown in SEQ ID NO. 1.
The chinese name of YGKRLWK is: tyrosine-glycine-lysine-arginine-leucine-tryptophan-lysine.
Further, the amino acid sequence of the polypeptide is X 1 -D-D-D-D-K 1 -R-X 2 -X 3 -K 2 ，
X 1 、X 2 、X 3 Is amino acid, D is aspartic acid, K 1 、K 2 Each is lysine and R is arginine;
wherein K is 1 R is enterokinase, lysyl endopeptidase, trypsin, double-base amino acid endopeptidase cleavage site, X 3 -K 2 Is carboxypeptidase B cleavage site;
preferably, the amino acid sequence of the polypeptide is YDDDDKRGWK.
X 1 -D-D-D-D-K 1 -R-X 2 -X 3 -K 2 As shown in SEQ ID NO. 2. YDDDDKRGWK is shown in SEQ ID NO. 3.
YDDDDKRGWK the Chinese name is: tyrosine-aspartic acid-lysine-arginine-glycine-tryptophan-lysine.
In another aspect, the invention provides a kit comprising the polypeptides for detecting residual amounts and activities of various proteases.
Further, the kit also comprises Tris-HCl buffer solution and Na + Salt, ca 2+ One or more of salt, ammonium bicarbonate and protease positive reference substances.
In yet another aspect of the present invention, there is provided the use of the above-described polypeptide or kit for detecting residual amounts and activities of various proteases in a biological product.
Further, the biologic includes insulin, GLP-1 receptor agonist drugs, and GLP-2 peptide analogs;
the protease comprises one or more of lysyl endopeptidase, trypsin, double-base amino acid endopeptidase, enterokinase and carboxypeptidase B.
In still another aspect of the present invention, a method for detecting residual amounts and activities of a plurality of proteases is provided, wherein the polypeptides for detecting residual amounts and activities of a plurality of proteases are used as substrates, and react with proteases in a sample to be detected, and the amount of decrease in substrate peptides or increase in product peptides is quantitatively analyzed, and the residual amounts and activities of proteases in the sample to be detected are calculated.
Further, the method comprises the following steps:
s1: preparing a sample solution to be tested and a polypeptide solution; when the protease residual quantity is detected, preparing a protease positive reference substance solution;
s2: mixing the polypeptide solution with a sample solution to be detected, and performing enzyme digestion reaction to obtain a sample reaction solution to be detected; when the protease residual quantity is detected, the method further comprises the steps of mixing a polypeptide solution, a sample solution to be detected and a protease positive reference substance solution, and performing enzyme digestion reaction to obtain a positive reference substance reaction solution;
s3: after the reaction is finished, carrying out high performance liquid chromatography detection on the reaction liquid of the sample to be detected; when the protease residual quantity is detected, the method also comprises the step of detecting the positive control reaction liquid by high performance liquid chromatography;
s4: calculating enzyme activity by detecting the decrease amount of the polypeptide or the increase amount of the enzyme digestion product in the reaction liquid of the sample to be detected; or detecting enzyme digestion products in the reaction liquid of the sample to be detected and the reaction liquid of the positive reference substance, and judging whether enzyme residues exist in the sample to be detected or not or whether the enzyme residues are smaller than the minimum limit value.
Further, the reaction temperature is 25-45 ℃, preferably 37 ℃; the reaction time is 10-20min; the pH of the reaction is 7.5-9.5;
preferably, the conditions for high performance liquid chromatography detection include:
chromatographic column: c18 chromatographic column, filler is octadecylsilane chemically bonded silica gel;
mobile phase a:0.1% aqueous tfa, mobile phase B:0.1% tfa acetonitrile;
detection wavelength: 214nm;
the flow rate is 0.6-1.2 ml/min;
gradient elution: the volume ratio of the mobile phase A to the mobile phase B is 95-5 percent, 35-65 percent or 90-10 percent, 70-30 percent.
The term "aromatic amino acid" in the present invention refers to an amino acid containing a benzene ring structure, and includes phenylalanine and its derivatives, tyrosine and its derivatives, tryptophan and its derivatives; "Heterocyclyl amino acid" refers to amino acids containing a heterocyclic structure, including histidine and derivatives thereof.
The technical principle of the invention is as follows: when detecting residual enzyme in the purified biological product, the conventional protease activity detection generally adopts a specific substrate of protease, such as Lys-PNA which is a substrate commonly adopted by lysyl endonuclease, and enzyme activity is calculated by detecting absorbance of an enzyme cleavage product after enzyme cleavage of the substrate due to specificity of enzyme catalytic reaction; when the biological product contains a plurality of residual enzymes, a plurality of substrates or measuring methods are required to be selected, and the operation is complex. In the experimental process, the inventor finds that through repeated experimental research and screening: the polypeptide substrate is suitable for detecting various residual enzymes, and has high efficiency and high detection sensitivity. When the protease is used for enzyme digestion of the polypeptide substrate, the substrate polypeptide and the product polypeptide can be effectively separated during detection by liquid chromatography, so that the content of the substrate polypeptide and the product polypeptide can be detected, and the peak area or the signal intensity and the content of the substrate polypeptide and/or the product polypeptide have a high linear relationship, so that various proteases and the content thereof can be detected simultaneously. This is probably because the polypeptide of the present invention includes not only endopeptidase cleavage site and exopeptidase cleavage site, but also amino acids having benzene ring or heterocyclic structure at specific positions, which not only improves cleavage efficiency, but also as signal enhancement site, improves the detection signal intensity of the polypeptide or cleavage product, thereby improving detection sensitivity.
Compared with the prior art, the invention has the following beneficial effects:
(1) The method has high sensitivity, the detection limit of the polypeptide is 0.03125mmol/L, and can detect various proteases at one time, and the detection limit of the protease can reach below mug level, thereby being beneficial to detecting the enzyme activity under the conditions of lower enzyme activity and fewer products.
(2) The polypeptide substrate of the invention has good universality and can rapidly detect various trace proteases (active forms).
(3) The invention has high specificity, the substrate peptide, the detected object and the product peptide show peaks at different times, do not interfere with each other, and are completely separated from the base line.
(4) The polypeptide substrate has small molecular weight and good stability.
(5) The method has the advantages of short digestion time, short time consumption of liquid chromatography or liquid mass spectrometry analysis, difficult blockage, contribution to rapid analysis of samples and high detection efficiency.
(6) The detection method has wide application range, and can be used for quantitative detection of enzyme activity and detection of various enzyme residues in protein medicines.
(7) The detection method is simple, easy to operate and suitable for industrial production.
Drawings
FIG. 1 is a linear fit of PJ-7S in example 2 of the present invention.
FIG. 2 is a linear fit of PJ-10S in example 2 of the present invention.
FIG. 3 is a liquid chromatogram of the rTrypsin cleavage PJ-7S of example 2 of the present invention.
FIG. 4 is a liquid chromatogram of rKex2 cleavage PJ-7S in example 2 of the present invention.
FIG. 5 is a liquid chromatogram of rCPB cleavage PJ-7S in example 2 of the present invention.
FIG. 6 is a liquid chromatogram of rLys-C cleavage PJ-7S in example 2 of the present invention.
FIG. 7 is a liquid chromatogram of rEK digested PJ-7S in example 2 of the invention.
FIG. 8 is a liquid chromatogram of a control group for detecting rLys-C enzyme residue in insulin de novo in example 3 of the present invention.
FIG. 9 is a liquid chromatogram of the experimental group for detecting the residue of rLys-C enzyme in insulin de novo in example 3 of the present invention.
FIG. 10 is a liquid chromatogram of the rCPB and rKex2 enzyme residue detection control group in the liraglutide intermediate of example 4 of the present invention.
FIG. 11 is a liquid chromatogram of the first batch of the test group for the detection of rCPB and rKex2 enzyme residues in the liraglutide intermediate of example 4 of the present invention.
FIG. 12 is a liquid chromatogram of a second batch of the test set for the detection of rCPB and rKex2 enzyme residues in the liraglutide intermediate of example 4 of the present invention.
FIG. 13 is a liquid chromatogram of a third run of the rCPB and rKex2 enzyme residue detection assay in liraglutide intermediate of example 4 of the present invention.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings and examples.
Example 1 preparation of the polypeptides PJ-7S and PJ-10S
The polypeptides PJ-7S and PJ-10S are prepared by adopting a chemical method to synthesize Jiangsu Jitai peptide, and the purity detection and content calibration are carried out, and the results are shown in Table 1:
TABLE 1 Polypeptides PJ-7S and PJ-10S
EXAMPLE 2 Activity assay of protease
1. Linear solution: PJ-7S was diluted 2-fold with 50mM Tris-HCl, pH8.0 to 6 concentrations below, 1mmol/L, 0.5mmol/L, 0.25mmol/L, 0.125mmol/L, 0.0625mmol/L, 0.03125mmol/L as standard curves. PJ-10S was diluted 2-fold with 50mM Tris-HCl, pH8.0, to 6 concentrations below, 0.5. Mu. Mol/L, 0.4. Mu. Mol/L, 0.3. Mu. Mol/L, 0.2. Mu. Mol/L, 0.1. Mu. Mol/L, 0.05. Mu. Mol/L as standard curves.
2. Enzyme digestion reaction
A certain amount of substrate peptide (PJ-7S and PJ-10S) is dissolved by different buffers for later use, protease with different concentrations is diluted to 1ug/ml, the protease is added into corresponding dissolution liquid at one time, 80ul (the addition amount of each protease is 0.08 ug) is respectively taken out after reaction for 10min at 37 ℃, and the protease is added into 20ul 1mol/L HCl to terminate enzyme digestion reaction to be detected. The types, amounts and corresponding substrate peptides and buffers of the proteases used are shown in Table 2.
TABLE 2 different polypeptides, buffers and protease loading tables
| Substrate peptide (1 mg/ml) | Dissolution buffering | Protease (1. Mu.g/ml) |
| PJ-7S | 50mM ammonium bicarbonate, 2mM Ca 2+ ，pH7.6～8.0 | rTrypsin |
| PJ-7S | 50～100mM Tris-HCl，2mM Ca 2+ ，pH 8.0～8.4 | rKex2 |
| PJ-7S | 50mM Tris-HCl，pH 7.8～8.2 | rCPB |
| PJ-10S | 50mM Tris-HCl，pH 8.8～9.2 | rLys-C |
| PJ-10S | 50mM Tris-HCl，pH 7.8～8.2 | rEK |
3. Detection conditions
The detection method comprises the following steps: a Welch XB-C18 4.6X100 mm,5 μm column was used, mobile phase: and (3) solution A:0.1% aqueous trifluoroacetic acid; and (2) liquid B:0.1% acetonitrile trifluoroacetic acid solution; wavelength 214nm flow rate: 1.0ml/min; gradient elution: the volume ratio of the mobile phase A to the mobile phase B is (95-5%): (35-65%) or (90-10%): (70-30%). Analysis was performed by injecting 20. Mu.l of sample, and a chromatogram was recorded.
4. Calculation of a Standard Curve
A standard curve is drawn according to the detection results of different concentrations of the PJ-7S and the PJ-10S polypeptides, and the results are shown in tables 3, 4, 1 and 2.
TABLE 3 statistical table of detection results for different concentrations of PJ-7S polypeptide
| Concentration mmol/L | 0 | 0.03125 | 0.0625 | 0.125 | 0.25 | 0.5 | 1 |
| Peak area | 0 | 824783 | 1728833 | 3525515 | 7329489 | 14509714 | 27852686 |
As can be seen from Table 3, the PJ-7S polypeptides have a good linear relationship in the concentration range of 0.03125-1 mmol/L, the fitting equation is y=3E+07x+89214, and the correlation coefficient R 2 ＝0.9995。
TABLE 4 statistical table of detection results for different concentrations of PJ-10S polypeptide
| Concentration mu mol/L | 0 | 0.05 | 0.1 | 0.2 | 0.3 | 0.4 | 0.5 |
| Peak area | 0 | 1575.7 | 3166.6 | 6310.0 | 9361.1 | 12308.2 | 15178.5 |
As can be seen from Table 4, the PJ-10S polypeptides have a good linear relationship in the concentration range of 0.05-0.5 mu mol/L, the fitting equation is y=304463+101.32, and the correlation coefficient R 2 ＝0.9997。
Calculating the substrate reduction: the peak area of the substrate peptide (non-cleaved polypeptide) in each reaction system is substituted into a standard curve formula, the residual amount of the substrate peptide (non-cleaved peptide fragment) is calculated, and the reduction amount is calculated according to the initial addition amount of the substrate peptide. The formula is as follows:
decrease = initial addition amount-residual amount
After cleavage, the corresponding cleavage product peptide Duan Feng and the uncleaved peptide Duan Feng can be found, and the maps are shown in fig. 3-7. The enzymatic activity of each protease can be obtained by calculation (methods of calculation refer to industry definitions or pharmacopoeia specifications).
Definition of enzyme activity unit: the amount of enzyme required to catalyze 1mol of substrate peptide per minute was 1 unit under cleavage buffer of each protease and reaction conditions of 37 ℃. The results are shown in Table 5:
TABLE 5 results of enzyme Activity assays
| Name of the name | Substrate peptides | Enzyme Activity (U/mg) |
| rTrypsin | PJ-7S | 200 |
| rKex2 | PJ-7S | 2 |
| rCPB | PJ-7S | 70 |
| rLys-C | PJ-10S | 20 |
| rEK | PJ-10S | 20 |
EXAMPLE 3 detection of rLys-C enzyme residue in De-Glutamine
Recombinant lysyl endopeptidase (abbreviated as rLys-C) is introduced into the production process of Degu insulin, so that the enzyme residue control of rLys-C is required.
A10 mg/ml solution of Degu insulin, a 1.0mg/ml solution of polypeptide (abbreviated as PJ-10S) and a 0.003AU/ml solution of rLys-C were prepared with 50mM Tris-HCl, pH8.0 buffer, and after each solution was added in this order according to Table 6, the reaction was carried out at 37℃for 10 minutes, 80. Mu.l of the sample was sampled, and 20. Mu.l of a 1mol/L HCl acetonitrile solution was added to terminate the cleavage.
TABLE 6 enzyme digestion and sample addition
| Grouping | PJ-10S(1.0mg/ml) | 50mM Tris-HCl, pH8.0 buffer | Degu (10 mg/ml) | rLys-C(0.003AU/ml) |
| Control group | 50μl | / | 50μl | 5μl |
| Experimental group | 50μl | 5μl | 50μl | / |
Samples were taken for HPLC analysis. The detection method comprises the following steps: a Welch XB-C18 4.6X100 mm,5 μm column was used, mobile phase: and (3) solution A:0.1% aqueous trifluoroacetic acid; and (2) liquid B:0.1% acetonitrile trifluoroacetic acid solution; wavelength 214nm gradient: within 0-40 min, the solution B is from 5-65%; flow rate: 1.0ml/min; analysis was performed by injecting 20. Mu.l. The chromatograms were recorded and the detection results are shown in table 7, fig. 8 and fig. 9.
TABLE 7 detection results of rLys-C enzyme residues in De-Glutamine
As is clear from the data in Table 7, the control group detected a newly increased peptide peak after cleavage by rLys-C in PJ-10S, whereas the experimental group did not detect the newly increased peptide peak, and CPB and Kex2 enzyme activity residual amounts were not detected. Calculated according to a limit check method, the residue of rLys-C enzyme in each mg of insulin deglutition is less than 3×10 -5 AU, is not exceeded by the limit specification.
Example 4 detection of rCPB and rKex2 enzyme residues in Liraglutide intermediate
Recombinant carboxypeptidase B (rCPB for short) and recombinant double basic amino acid endopeptidase (rKex 2 for short) are introduced into the production process of the liraglutide intermediate, so that enzyme residue control is required for rCPB and rKex 2.
20mg/ml of liraglutide intermediate solution, 1.0mg/ml of PJ-7S solution, 0.15U/ml of rCPB solution, 0.01U/ml of rKex2 solution were prepared with 50mM Tris-HCl buffer at pH8.0, and after each solution was added in this order according to Table 8, after 10 minutes of reaction at 37℃80. Mu.l was sampled and 20. Mu.l of 1mol/L HCl acetonitrile solution was added to terminate the cleavage.
TABLE 8 enzyme digestion and sample addition
Samples were taken for HPLC analysis. The detection method comprises the following steps: a Welch XB-C18 4.6X100 mm,5 μm column was used, mobile phase: and (3) solution A:0.1% aqueous trifluoroacetic acid; and (2) liquid B:0.1% acetonitrile trifluoroacetic acid solution; wavelength 214nm gradient: within 0-20min, the solution B is from 5-65%; flow rate: 1.0ml/min; analysis was performed by injecting 20. Mu.l. The chromatograms were recorded, and the detection results are shown in table 9, fig. 10 to fig. 13.
TABLE 9 results of detection of rCPB, rKex2 enzyme residues in liraglutide intermediates
As can be seen from the data in Table 9, the control group detected a new increased peptide peak after the PJ-7S was digested with rCPB and rKex2, the experimental group did not detect a new increased peptide peak, and CPB and Kex2 enzyme activity residues were not detected in all three batches of liraglutide intermediates. According to the calculation of a limit check method, three batches of liraglutide intermediates are all with enzyme activities corresponding to rCPB in each mg of liraglutide intermediates lower than 3.5x10 -6 U, rKex2 enzyme activities are all lower than 2.5x10 -7 U, not exceeding the limit.
EXAMPLE 5 detection of rCPB and rKex2 enzyme residues in cable Ma Lutai
Recombinant carboxypeptidase B (rCPB for short) and recombinant double basic amino acid endopeptidase (rKex 2 for short) are introduced into the production process of the Somar rupeptide, so that enzyme residue control is required for the rCPB and the rKex 2.
And (3) adopting PJ-10S as a substrate to control enzyme residues. Wherein the rCPB cleaves PJ-10S to produce a peptide fragment: YDDDDKRGW (code CW-9S); cleavage of PJ-10S by rKex2 resulted in peptide fragments: YDDDKR (code CW-7S) and GWK (code CW-3S). Polypeptides CW-7S, CW-3S and CW-9S were prepared by chemical synthesis of Jiangsu Jitai peptide, and were subjected to purity detection and content calibration, the results are shown in Table 10:
TABLE 10 information table of synthetic polypeptides
| SEQ ID NO.3 | SEQ ID NO.4 | SEQ ID NO.5 | |
| (Code) | CW-7S | CW-9S | CW-3S |
| Amino acid sequence | YDDDDKR | YDDDDKRGW | GWK |
| Purity of | 99.5％ | 99.2％ | 99.2％ |
| Content of | 2.2 mg/branch | 1.8 mg/branch | 2.1 mg/branch |
A solution of 20mg/ml of the cable-stuffiness peptide, a solution of 1.0mg/ml of PJ-7S, a solution of 0.15U/ml of rCPB and a solution of 0.01U/ml of rKex2 were prepared with 50mM Tris-HCl and pH8.0 buffer, and after each solution was added in this order according to Table 10, the reaction was carried out at 37℃for 10 minutes, 80. Mu.l of the sample was sampled and 20. Mu.l of a 1mol/L HCl solution was added to terminate the cleavage.
Quality control group: polypeptides CW-7S, CW-3S and CW-9S were prepared at final concentrations of 0.1mmol/L in 50mM Tris-HCl, pH8.0 buffer, 80. Mu.l of sample was taken, 20. Mu.l of 1mol/L HCl solution was added and mixed well, and samples were taken for HPLC analysis.
TABLE 11 enzyme digestion and sample addition amount
Samples were taken for HPLC analysis. The detection method comprises the following steps: a Welch XB-C18 4.6X100 mm,5 μm column was used, mobile phase: and (3) solution A:0.1% aqueous trifluoroacetic acid; and (2) liquid B:0.1% acetonitrile trifluoroacetic acid solution; wavelength 214nm gradient: within 0-20min, the solution B is from 5-65%; flow rate: 1.0ml/min; analysis was performed by injecting 20. Mu.l. The chromatogram was recorded and the detection results are shown in Table 12.
TABLE 12 detection results of rCPB, rKex2 enzyme residues in Somarlutide
As can be seen from the data in Table 12, the control group detected a new increased peptide peak after the PJ-10S was digested with rCPB and rKex2, the experimental group did not detect a new increased peptide peak, and the residual amounts of CPB and Kex2 enzyme activities were not detected in the three batches of the sorux peptides. According to the calculation of a limit check method, three batches of the somalupeptide are respectively lower than 3.5x10 in enzyme activity corresponding to rCPB in each mg of the somalupeptide -6 U, rKex2 enzyme activities are all lower than 2.5x10 -7 U, not exceeding the limit.
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Claims (10)
1. A polypeptide for detecting the residual and activity of a plurality of proteases, characterized in that: the polypeptide comprises at least one enzyme cutting site, and the number of amino acids contained in the polypeptide is a,3< a <20; at least one of the amino acids is one of aromatic amino acid and derivatives thereof, heterocyclic amino acid and derivatives thereof and is positioned at the N end or the C end of the enzyme cutting site; the enzyme cutting sites comprise one or more of endopeptidase cutting sites and exopeptidase cutting sites;
preferably, the aromatic amino acids include tyrosine, tryptophan, phenylalanine; the heterocyclic amino acid includes histidine; the endopeptidase comprises lysyl endopeptidase, trypsin, pepsin, enterokinase, double-base amino acid endopeptidase, chymotrypsin; the exopeptidase includes carboxypeptidase and aminopeptidase.
2. The polypeptide for detecting the residual quantity and the activity of a plurality of proteases according to claim 1, wherein: the amino acid sequence of the polypeptide is X 1 -X 2 -K 1 -R-X 3 -X 4 -K 2 ，
X 1 、X 2 、X 3 、X 4 Is amino acid, K 1 、K 2 Each is lysine and R is arginine;
wherein X is 2 -K 1 K is a cleavage site of lysyl endopeptidase and trypsin 1 R is a double-base amino acid endopeptidase, trypsin cleavage site, X 4 -K 2 Is carboxypeptidase B cleavage site;
preferably, the amino acid sequence of the polypeptide is YGKRLWK.
3. The polypeptide for detecting the residual quantity and the activity of a plurality of proteases according to claim 1, wherein: the amino acid sequence of the polypeptide is X 1 -D-D-D-D-K 1 -R-X 2 -X 3 -K 2 ，
X 1 、X 2 、X 3 Is amino acid, D is aspartic acid, K 1 、K 2 Each is lysine and R is arginine;
wherein K is 1 R is enterokinase, lysyl endopeptidase, trypsin, double-base amino acid endopeptidase cleavage site, X 3 -K 2 Is carboxypeptidase B cleavage site;
preferably, the amino acid sequence of the polypeptide is YDDDDKRGWK.
4. A kit comprising the polypeptide of any one of claims 1-3 for detection of residual amounts and activities of a plurality of proteases.
5. The kit of claim 4, further comprising Tris-HCl buffer, na + Salt, ca 2+ One or more of salt, ammonium bicarbonate and protease positive reference substances.
6. Use of a polypeptide for detecting residual amounts and activities of a plurality of proteases according to any one of claims 1 to 3, a kit according to claim 4 or 5 for detecting residual amounts and activities of proteases in biological products.
7. The use according to claim 6, characterized in that: the biological product comprises insulin, GLP-1 receptor agonist drug, GLP-2 peptide analogue;
the protease comprises one or more of lysyl endopeptidase, trypsin, double-base amino acid endopeptidase, enterokinase and carboxypeptidase B.
8. A method for detecting residual amounts and activities of a plurality of proteases, comprising: taking the polypeptide for detecting the residual quantity and the activity of various proteases as a substrate, reacting with the proteases in a sample to be detected, quantitatively analyzing the reduced quantity of the substrate peptide or the increased quantity of the product peptide, and calculating to obtain the residual quantity and the activity of the proteases in the sample to be detected.
9. The method for detecting the residual amounts and activities of a plurality of proteases according to claim 8, wherein: the method comprises the following steps:
s1: preparing a sample solution to be tested and a polypeptide solution; when the protease residual quantity is detected, preparing a protease positive reference substance solution;
s2: mixing the polypeptide solution with a sample solution to be detected, and performing enzyme digestion reaction to obtain a sample reaction solution to be detected; when the protease residual quantity is detected, the method further comprises the steps of mixing a polypeptide solution, a sample solution to be detected and a protease positive reference substance solution, and performing enzyme digestion reaction to obtain a positive reference substance reaction solution;
s3: after the reaction is finished, carrying out high performance liquid chromatography detection on the reaction liquid of the sample to be detected; when the protease residual quantity is detected, the method also comprises the step of detecting the positive control reaction liquid by high performance liquid chromatography;
s4: calculating enzyme activity by detecting the decrease amount of the polypeptide or the increase amount of the enzyme digestion product in the reaction liquid of the sample to be detected; or detecting enzyme digestion products in the reaction liquid of the sample to be detected and the reaction liquid of the positive reference substance, and judging whether enzyme residues exist in the sample to be detected or not or whether the enzyme residues are smaller than the minimum limit value.
10. The method for detecting the residual amounts and activities of a plurality of proteases according to claim 9, wherein: the reaction temperature is 25-45 ℃, preferably 37 ℃; the reaction time is 10-20min; the pH of the reaction is 7.5-9.5;
preferably, the conditions for high performance liquid chromatography detection include:
chromatographic column: c18 chromatographic column, filler is octadecylsilane chemically bonded silica gel;
mobile phase a:0.1% aqueous tfa, mobile phase B:0.1% tfa acetonitrile;
detection wavelength: 214nm;
the flow rate is 0.6-1.2 ml/min;
gradient elution: the volume ratio of the mobile phase A to the mobile phase B is 95-5 percent, 35-65 percent or 90-10 percent, 70-30 percent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310562097.4A CN116804041A (en) | 2023-05-18 | 2023-05-18 | Polypeptide for detecting residual quantity and activity of various proteases and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310562097.4A CN116804041A (en) | 2023-05-18 | 2023-05-18 | Polypeptide for detecting residual quantity and activity of various proteases and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116804041A true CN116804041A (en) | 2023-09-26 |
Family
ID=88080105
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310562097.4A Pending CN116804041A (en) | 2023-05-18 | 2023-05-18 | Polypeptide for detecting residual quantity and activity of various proteases and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116804041A (en) |
-
2023
- 2023-05-18 CN CN202310562097.4A patent/CN116804041A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| St. Hilaire et al. | Fluorescence-quenched solid phase combinatorial libraries in the characterization of cysteine protease substrate specificity | |
| Appel | Chymotrypsin: molecular and catalytic properties | |
| Dall'Acqua et al. | Substrate-assisted catalysis: molecular basis and biological significance | |
| US8669117B2 (en) | Method for single oxygen atom incorporation into peptides | |
| Péter et al. | Liquid chromatographic study of the enzymatic degradation of endomorphins, with identification by electrospray ionization mass spectrometry | |
| Temporini et al. | Optimization of a trypsin-bioreactor coupled with high-performance liquid chromatography–electrospray ionization tandem mass spectrometry for quality control of biotechnological drugs | |
| CN113164557A (en) | Methods of treating neurological disorders | |
| Wang et al. | A continuous colorimetric assay for rhinovirus-14 3C protease using peptidep-nitroanilides as substrates | |
| Gopi et al. | Proteolytic stability of β-peptide bonds probed using quenched fluorescent substrates incorporating a hemoglobin cleavage site | |
| CN116804041A (en) | Polypeptide for detecting residual quantity and activity of various proteases and application thereof | |
| Le et al. | A sensitive HPLC method for measuring bacterial proteolysis and proteinase activity in UHT milk | |
| US8609353B2 (en) | Diagnostics and methods for removal and detection of interferents | |
| CN1325455A (en) | Enzymatic amidation of peptides | |
| da Silva Gomes et al. | A fluorimetric method for the determination of pepsin activity | |
| CN115808484B (en) | Method for detecting residual lysyl endonuclease in insulin or insulin analogue | |
| US20190004036A1 (en) | Methods, compositions, and kits for detection of bacterial proteases | |
| Thompson et al. | Analysis of conserved residues of the human puromycin-sensitive aminopeptidase | |
| US8580534B2 (en) | Method for incorporation of two oxygen atoms into digested peptides using peptidases | |
| CN116046947B (en) | Method for detecting residual lysyl endonuclease in insulin diglucer | |
| US20160053248A1 (en) | Systems and methods for evolving enzymes with desired activities | |
| Llerena-Suster et al. | Selective adsorption of plant cysteine peptidases onto TiO2 | |
| JP2006516900A (en) | Improved method for the detection of proteolytic enzymes | |
| SR et al. | Substrate specificity profiling of peptidyl-Lys metallopeptidase of Armillaria mellea by FRET based peptide library | |
| Mima et al. | Overexpression and functional characterization of a serine carboxypeptidase inhibitor (IC) from Saccharomyces cerevisiae | |
| EP2350310A1 (en) | High-throughput assay for evaluating dipeptidyl peptidase i activity |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |