Variants of porcine trypsin

Patent metadata

Field	Value
Publication number	US-11952602-B2
Application number	US-202218050446-A
Country	US
Kind code	B2
Filing date	Oct 27, 2022
Priority date	Feb 22, 2018
Publication date	Apr 9, 2024
Grant date	Apr 9, 2024

Abstract

The present invention relates to polypeptide variants of porcine trypsin, to nucleic acid molecules encoding these variants, and to host cells comprising such nucleic acid molecules. It also relates to the use of these variants in methods for producing insulin. The invention further relates to the use of these variants as medicaments, as food ingredients, or as feed ingredients and to the use of these variants within a process of manufacturing a food ingredient or a feed ingredient.

First claim

The invention claimed is:

1. A method for the production of human insulin, an insulin analogue or a derivative of insulin, the method comprising using a variant of porcine trypsin, wherein the variant of porcine trypsin comprises:
an amino acid sequence that has at least 91% sequence identity to SEQ ID NO: 1,
wherein said amino acid sequence differs from SEQ ID NO: 1 at least by one or more amino acid substitutions at one or more positions corresponding to F24, S44, D56, G78, Y131, S172 and W193 of native porcine trypsin according to SEQ ID NO: 1,
with the proviso that said amino acid sequence is not native porcine trypsin according to SEQ ID NO: 1; and
with the proviso that said amino acid sequence is not porcine variant trypsin S172A according to SEQ ID NO: 2.

2. The method of claim 1 , wherein the insulin analogue is selected from the group consisting of insulin aspart, insulin lispro, insulin glulisine, and insulin glargine.

3. The method of claim 1 , wherein the amino acid at the position corresponding to F24 is substituted by an amino acid selected from the group consisting of Ala, Asn, Arg, Gln, Ile, Leu, Lys, Met, Ser, Thr, and Val.

4. The method of claim 1 , wherein the amino acid at the position corresponding to S44 is substituted by an amino acid selected from the group consisting of Leu and Pro.

5. The method of claim 1 , wherein the amino acid at the position corresponding to D56 is substituted by an amino acid selected from the group consisting of Ala, Asn, His, and Trp.

6. The method of claim 1 , wherein the amino acid at the position corresponding to G78 is substituted by an amino acid selected from the group consisting of Ala, Glu; Pro, Ser, and Tyr.

7. The method of claim 1 , wherein the amino acid at the position corresponding to Y131 is substituted by an amino acid selected from the group consisting of Ala, Asn, Asp, Cys, Gln; Glu, Gly, His, Ile, Leu, Met, Ser, Thr, Trp, and Val.

8. The method of claim 1 , wherein the amino acid at the position corresponding to S172 is substituted by an amino acid selected from the group consisting of Ala, Cys, and Thr.

9. The method of claim 1 , wherein the amino acid at the position corresponding to W193 is substituted by an amino acid selected from the group consisting of Phe, Ser, Thr, and Tyr.

10. The method of claim 1 , wherein said amino acid sequence additionally differs from SEQ ID NO: 1 at least by one or more amino acid substitutions at one or more positions corresponding to R99, R107, K125, and K170 of native porcine trypsin according to SEQ ID NO: 1.

11. The method of claim 10 , wherein the amino acid at the position corresponding to R99 is substituted by an amino acid selected from the group consisting of Ala, Asn, Asp, Glu, Gly, His, Leu, Phe, Thr, Trp, and Tyr.

12. The method of claim 10 , wherein the amino acid at the position corresponding to R107 is substituted by an amino acid selected from the group consisting of Asp, Gly, Pro, Ser, and Thr.

13. The method of claim 10 , wherein the amino acid at the position corresponding to K125 is substituted by an amino acid selected from the group consisting of Ala, Cys, Gln, Glu, Gly, His, Leu, Ser, and Tyr.

14. The method of claim 10 , wherein the amino acid at the position corresponding to K170 is substituted by an amino acid selected from the group consisting of Ala, Asn, Gly, and Tyr.

15. The method of claim 1 , wherein said amino acid sequence is SEQ ID NO: 3, wherein Xaa24 is an amino acid selected from the group consisting of Ala, Asn, Arg, Gln, Ile, Leu, Lys, Met, Phe, Ser, Thr, and Val, with the proviso that SEQ ID NO: 3 is not the sequence of SEQ ID NOs: 1 and 2.

16. The method of claim 1 , wherein said amino acid sequence is SEQ ID NO: 3, wherein
Xaa44 is an amino acid selected from the group consisting of Leu, Pro, and Ser;
Xaa56 is an amino acid selected from the group consisting of Ala, Asn, Asp, His, and Trp;
Xaa78 is an amino acid selected from the group consisting of Ala, Glu, Gly, Pro, Ser, and Tyr;
Xaa99 is an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Glu, Gly, His, Leu, Phe, Thr, Trp, and Tyr;
Xaa107 is an amino acid selected from the group consisting of Arg, Asp, Gly, Pro, Ser, and Thr;
Xaa125 is an amino acid selected from the group consisting of Ala, Cys, Gln, Glu, Gly, His, Leu, Lys, Ser, and Tyr;
Xaa131 is an amino acid selected from the group consisting of Ala, Asn, Asp, Cys, Gln; Glu, Gly, His, Ile, Leu, Met, Ser, Thr, Trp, Tyr, and Val;
Xaa170 is an amino acid selected from the group consisting of Ala, Asn, Gly, Lys, and Tyr;
Xaa172 is an amino acid selected from the group consisting of Ala, Cys, Ser, and Thr; and/or
Xaa193 is an amino acid selected from the group consisting of Phe, Ser, Thr, Trp, and Tyr; with the proviso that SEQ ID NO: 3 is not the sequence of SEQ ID NOs: 1 and 2.

17. The method of claim 1 , wherein said amino acid sequence is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO:, 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, and SEQ ID NO: 119.

18. The method of claim 1 , wherein said variant is capable of cleaving a peptide with the general formula A-Lys-Thr-Arg-Arg-B according to SEQ ID NO: 131 to yield a cleavage product of the general formula A-Lys-Thr-Arg-Arg according to SEQ ID NO: 132 in a yield of at least 80%,
wherein A is an amino acid sequence consisting of one or more amino acids; and
wherein B is an amino acid sequence consisting of one or more amino acids.

19. The method of claim 1 , wherein the variant exhibits an increased selectivity for the cleavage of a pre-pro-human insulin, pre-pro-insulin glargine, pre-pro-insulin lispro, pre-pro-insulin aspart, or pre-pro-insulin glulisine at a position C-terminally to position B32-Arg as compared to porcine variant trypsin S172A according to SEQ ID NO: 2.

20. The method of claim 1 , further comprising contacting a pre-pro

Assignees

• Sanofi Aventis Deutschland

Inventors

• Stillger Thomas
• Rissom Sebastian
• Feller Claudia
• Vogel Andreas

Classifications

• C12N9/6427 Primary

  Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4) · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12Y304/21004 Primary

  Trypsin (3.4.21.4) · CPC title

• C07K14/62

  Insulins · CPC title