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US-20260125664-A1 - COMPOSITIONS AND METHODS RELATING TO RECOMBINANT CHYMOTRYPSIN

US20260125664A1US 20260125664 A1US20260125664 A1US 20260125664A1US-20260125664-A1

Abstract

Provided herein are compositions, methods, and kits related to chymotrypsin production for characterizing proteins, assessing, and monitoring disease stages and phases, predicting the likelihood of disease progression, predicting and monitoring responses to disease therapies, and treating disease conditions.

Inventors

  • Alba Katiria GONZALEZ RIVERA
  • Sergei Saveliev
  • Marjeta Urh
  • Michael Killoran
  • Lance Encell

Assignees

  • PROMEGA CORPORATION

Dates

Publication Date
20260507
Application Date
20250730

Claims (17)

  1. 1 - 144 . (canceled)
  2. 145 . A composition comprising: (a) a cell comprising an exogenous nucleic acid sequence encoding a recombinant chymotrypsin polypeptide comprising at least 70% sequence identity to SEQ ID NO: 32 or 33; (b) a nucleic acid sequence encoding a recombinant chymotrypsin polypeptide comprising at least 70% sequence identity to SEQ ID NO: 32 or 33; or (c) a recombinant chymotrypsin polypeptide comprising at least 70% sequence identity to SEQ ID NO: 32 or 33.
  3. 146 . The composition of claim 145 , wherein the nucleic acid sequence is stably integrated into the genome of the cell.
  4. 147 . The composition of claim 145 , wherein the nucleic acid sequence is transiently expressed in the cell.
  5. 148 . The composition of claim 145 , wherein the cell is a yeast cell.
  6. 149 . The composition of claim 148 , wherein the cell is Pichia pastoris.
  7. 150 . The composition of claim 145 , wherein the recombinant chymotrypsin polypeptide comprises one or more of a secretion signal, a pro-peptide, or a purification tag.
  8. 151 . The composition of claim 145 , wherein the exogenous nucleic acid sequence further comprises a promoter and/or encodes any one or more of a secretion signal, a pro-peptide, or a purification tag.
  9. 152 . The composition of claim 151 , wherein the purification tag is a C-terminal histidine tag.
  10. 153 . The composition of claim 145 , wherein the exogenous nucleic acid sequence comprises at least 70% sequence identity to SEQ ID NO: 105 or 106.
  11. 154 . The composition of claim 145 , wherein the mutant honeybee recombinant chymotrypsin comprises one or more of a S at position 180, a C at position 191, a DI at position 204, and a V at position 211 relative to SEQ ID NO: 34.
  12. 155 . The composition of claim 145 , wherein the recombinant chymotrypsin polypeptide further comprises at least 70% sequence identity to SEQ ID NO: 20.
  13. 156 . The composition of claim 145 , wherein the recombinant chymotrypsin polypeptide further comprises at least 70% sequence identity to SEQ ID NO: 21.
  14. 157 . The composition of claim 145 , wherein the recombinant chymotrypsin polypeptide comprises at least 90% sequence identity to SEQ ID NO 32 or 33.
  15. 158 . The composition of claim 157 , wherein the recombinant chymotrypsin polypeptide comprises 100% sequence identity to SEQ ID NO 32 or 33.
  16. 159 . The composition of claim 145 , wherein the recombinant chymotrypsin polypeptide comprises 100% sequence identity to SEQ ID NO: 20.
  17. 160 . The composition of claim 145 , wherein the recombinant chymotrypsin polypeptide comprises at least 100% sequence identity to SEQ ID NO: 21.

Description

CROSS-REFERENCE This application claims the benefit of U.S. Provisional Patent Application No. 63/677,360, filed Jul. 30, 2024; which is incorporated by reference herein in its entirety. SEQUENCE LISTING The text of the computer readable sequence listing filed herewith, titled “PRMG_43026_202_SequenceListing_corrected.xml,” created Sep. 29, 2025, having a file size of 340,809 bytes, is hereby incorporated by reference in its entirety. FIELD Provided herein are compositions, methods, and kits related to recombinant chymotrypsin production for protein analytical applications such as peptide mapping and proteomics and clinical use including treatment of wounds, ulcers, asthma, and other applications. BACKGROUND Chymotrypsin can be used to characterize proteins. However, their use is limited due to their compromised cleavage specificity (e.g., chymotrypsins can cleave at aromatic residues tyrosine, phenylalanine, and tryptophan, but also cleave at other residues, which complicates protein analysis), protein analysis reproducibility (e.g., reproducibility of protein analysis using chymotrypsin is adversely affected by the age and health of the animal from which it is extracted), and performance (e.g., performance is compromised by contamination with proteases that are co-extracted with chymotrypsin from pancreas). Thus, compositions and methods are needed for chymotrypsin production with improved cleavage specificity, high lot-to-lot reproducibility and straightforward enzyme purification and improved purity. SUMMARY Provided herein are compositions, methods, and kits related to chymotrypsin production for protein analytical applications such as peptide mapping and proteomics and clinical use including treatment of wounds, ulcers, asthma, and other applications. Embodiments of the present disclosure include compositions comprising an exogenous nucleic acid sequence encoding a recombinant chymotrypsin polypeptide. In some embodiments, the nucleic acid sequence is stably integrated into the genome of a cell. In some embodiments, the nucleic acid sequence is transiently expressed in a cell. In some embodiments, the cell is a yeast cell. In some embodiments, the cell is Pichia pastoris. In some embodiments, the recombinant chymotrypsin polypeptide comprises any one or more of a secretion signal, a pro-peptide, or a purification tag. In some embodiments, the exogenous nucleic acid sequence comprises a promoter and/or encodes any one or more of a secretion signal, a pro-peptide, or a purification tag. Embodiments of the present disclosure include compositions comprising a recombinant chymotrypsin polypeptide. In some embodiments, the recombinant chymotrypsin polypeptide is expressed from a nucleic acid sequence that is stably or transiently integrated into the genome of a cell. In some embodiments, the cell is a yeast cell. In some embodiments, the cell is Pichia pastoris. In some embodiments, the recombinant chymotrypsin polypeptide comprises any one or more of a secretion signal, a pro-peptide, or a purification tag. In some embodiments, the recombinant chymotrypsin polypeptide and/or polynucleotide encoding a c recombinant chymotrypsin polypeptide comprises a sequence of any one of a Bovine (Bos taurus) chymotrypsin, a Porcine (Sus scrofa) chymotrypsin B, a Porcine (Sus scrofa) chymotrypsin variant N127Q, a Cod (Gadus morhua) chymotrypsin A, a Cod (Gadus morhua) chymotrypsin B, a Zebrafish (Danio rerio) chymotrypsin, a Beauveria bassiana chymotrypsin variant S81L, a Snake (Daboia siamensis) chymotrypsin E5L0E4 variant T62R/N81S/N83S/N134Q, a Snake (Macrovipera lebetinus) chymotrypsin VLCTLP, a Snake (Macrovipera lebetinus) chymotrypsin VLCTLP variant N26Q/N82Q/N98Q/N135Q/N232Q, a Snake (Macrovipera lebetinus) chymotrypsin VLCTLP variant N26Q/N82Q/N98Q/N135Q/N232A, a Snake (Macrovipera lebetinus) chymotrypsin VLAF, a Snake (Macrovipera lebetinus) chymotrypsin VLAF variant N26Q/N61Q/N83Q, a Goat (Capra hircus) chymotrypsin, a Rat (Rattus norvegicus) chymotrypsin, a Rabbit (Oryctolagus cuniculus) chymotrypsin, a Whale (Balaenoptera acutorostrata scammoni) chymotrypsin, a Chicken (Gallus gallus) chymotrypsin, a Crocodile (Crocodylus porosus) chymotrypsin, a Bumble bee (Bombus vosnesenskii) chymotrypsin, a Asian honeybee (Apis cerena cerena) chymotrypsin, an Ant (Solenopsis invicta) chymotrypsin, a Butterfly (Danaus plexippus Plexippus) chymotrypsin, a Firefly (Photinus pyralis) chymotrypsin, a Mosquito (Aedes aegypti) chymotrypsin, a Beetle (Tenebrio molitor) chymotrypsin A0A8J6LPH0, a Beetle (Tenebrio molitor) chymotrypsin A0A8J6HC31, a Beetle (Tenebrio molitor) chymotrypsin A0A8J6GYN2, a Moth (Diatraea saccharalis) chymotrypsin A0A9N9R2F0, a Honeybee (Apis mellifera) chymotrypsin L3W4, a Honeybee (Apis mellifera) chymotrypsin R3J2, a Honeybee (Apis mellifera) chymotrypsin, a Honeybee (Apis mellifera) chymotrypsin variant A180S/V191C/P204DI/D211V, a Honeybee (Apis mellifera) chymotrypsin variant C127A, a Honeybee (Api