IP Library › Granted Patent US 8,394,941
Granted Patent B2
US 8,394,941 · App. 13/082,979 · Granted Mar 12, 2013

Modified variant Bowman Birk Protease Inhibitors

Inventors: Neelam S. Amin (Burlingame, CA); Katherine D. Collier (Los Altos, CA); Melodie Estabrook (Mountian View, CA); David A. Estell (San Francisco, CA); Bryan P. Fox (Pacifica, CA); Scott D. Power (San Bruno, CA); Brian F. Schmidt (Half Moon Bay, CA); Gudrun Vogtentanz (Leiden, NL)
Assignee: Danisco US Inc.
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Quick Facts
Patent No.
US 8,394,941
App. No.
13/082,979
Granted
Mar 12, 2013
Kind
B2
Abstract

The present invention relates to modified variant Bowman Birk Protease Inhibitor proteins (BBPIs) that comprise peptides that bind target proteins, and that are further modified to have greater protease inhibitory activity and/or be produced at greater yields than the unmodified BBPIs. The invention encompasses polynucleotide constructs and expression vectors containing polynucleotide sequences that encode the modified variant BBPIs, the transformed host cells that express and produce the modified variant BBPIs, the modified variant BBPI proteins, the compositions comprising the modified variant BBPIs, and the methods for making and using the modified variant BBPIs in personal care.

Claims (21)

1. An isolated polynucleotide encoding a Bowman Birk Protease Inhibitor (BBPI) fusion protein, said polynucleotide comprising a first polynucleotide sequence encoding the catalytic unit of an enzyme, and a second polynucleotide sequence encoding a modified variant BBPI chosen from a VEGF-binding BBPI (VEGF-BBPI), an FGF-binding BBPI (FGF-BBPI), a TGF-binding BBPI (TGF-BBPI) and a TNF-binding BBPI (TNF-BBPI), wherein the backbone of the modified variant BBPI is substituted at one or more amino acid positions selected from the group consisting of positions equivalent to 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of SEQ ID NO:187, wherein the substitution at position 1 is to A or C, the substitution at position 4 is to V, the substitution at position 5 is to P or A, the substitution at position 11 is to G, the substitution at position 13 is to Y, I, F, M, L, V, K or R, the substitution at position 18 is to I, V or L, the substitution at position 25 is to K, N, W, I, A, R or L, the substitution at position 27 is to R, K, V, A or Q, the substitution at position 29 is to R, K, P or E, the substitution at position 31 is to Q, H, E, A, R, W, K or T, the substitution at position 38 is to N, K or R, the substitution at position 40 is to H, K, Q, R or Y, the substitution at position 50 is to R, Q, K, T, V, M or S, the substitution at position 52 is to K, T, R, Q, L, H, A, M, S or E, the substitution at position 55 is to M and the substitution at position 65 is to E, Q or D.

2. The isolated polynucleotide of claim 1 , wherein said first sequence encodes the catalytic unit of a cellulase.

3. An expression vector comprising the polynucleotide encoding the BBPI fusion protein of claim 1 .

4. The expression vector of claim 3 , wherein the second protease inhibitory loop of the modified variant BBPI of said BBPI fusion protein is a binding peptide chosen from a VEGF binding peptide, an FGF5 binding peptide, a TGFβ binding peptide and a TNFα binding peptide.

5. A host cell transformed with the vector of claim 3 .

6. The host cell of claim 5 , wherein said host cell is a Bacillus species host cell.

7. The polynucleotide of claim 1 , wherein the second protease inhibitory loop of said modified variant BBPI of said BBPI fusion protein is a binding peptide chosen from a VEGF binding peptide, an FGF5 binding peptide, a TGFβ binding peptide and a TNFα binding peptide.

8. The polynucleotide of claim 7 , wherein said binding peptide is a VEGF binding peptide selected from the group consisting of: YNLYGWT (SEQ ID NO: 676), ACYNLYGWTC (SEQ ID NO:9), KYYLYWW (SEQ ID NO:458), TLWKSYW (SEQ ID NO:459), DLYWW (SEQ ID NO:460), SKHSQIT (SEQ ID NO:468) KTNPSGS (SEQ ID NO:469) RPTGHSL (SEQ ID NO:470), KHSAKAE (SEQ ID NO:471) KPSSASS (SEQ ID NO:472), PVTKRVH (SEQ ID NO:473), TLHWWVT (SEQ ID NO:492), PYKASFY (SEQ ID NO:493), PLRTSHT (SEQ ID NO:494), EATPROT (SEQ ID NO:495), NPLHTLS (SEQ ID NO:496), KHERIWS (SEQ ID NO:497), ATNPPPM (SEQ ID NO:498), STTSPNM (SEQ ID NO:499), ADRSFRY (SEQ ID NO:500), PKADSKQ (SEQ ID NO:501), PNQSHLH (SEQ ID NO:502), SGSETWM (SEQ ID NO:503), ALSAPYS (SEQ ID NO:504), KMPTSKV (SEQ ID NO:505), ITPKRPY (SEQ ID NO:506), KWIVSET (SEQ ID NO:507), PNANAPS (SEQ ID NO:508), NVQSLPL (SEQ ID NO:509), TLWPTFW (SEQ ID NO:510), NLWPHFW (SEQ ID NO:511), SLWPAFW (SEQ ID NO:512), SLWPHFW (SEQ ID NO:513), APWNSHI (SEQ ID NO:514), APWNLHI (SEQ ID NO:515), LPSWHLR (SEQ ID NO:516), PTILEWY (SEQ ID NO:517), TLYPQFW (SEQ ID NO:518), HLAPSAV (SEQ ID NO:519), KYYLSWW (SEQ ID NO:520), WYTLYKW (SEQ ID NO:521), TYRLYWW (SEQ ID NO:522), RYSLYYW (SEQ ID NO:523), YYLYYWK (SEQ ID NO:524), NYQLYGW (SEQ ID NO:525), TKWPSYW (SEQ ID NO:226), TLWKSYW (SEQ ID NO:527), PLWPSYW (SEQ ID NO:528), RLWPSYW (SEQ ID NO:529), TLWPKYW (SEQ ID NO:530), KYDLYWW (SEQ ID NO: 531), RYDLYWW (SEQ ID NO:532), DYRLYWW (SEQ ID NO:533), DYKLYWW (SEQ ID NO:534), EYKLYWW (SEQ ID NO:535), and RYPLYWW (SEQ ID NO:536).

9. The polynucleotide of claim 7 , wherein said binding peptide is an FGF-5 binding peptide selected from the group consisting of: CACRTQPYPLCF (MM007; SEQ ID NO:430), CICTWIDSTPC (PS2; SEQ ID NO:431), CYGLPFTRC (SEQ ID NO:537), CEEIWTMLC (SEQ ID NO:538), CWALTVKTC (SEQ ID NO:539), CLTVLWTTC (SEQ ID NO:540), CTLWNRSPC (SEQ ID NO:541), CHYLLTNYC (SEQ ID NO:542), CRIHLAHKC (SEQ ID NO:543), TNIDSTP (SEQ ID NO:544), HLQTTET (SEQ ID NO:545), SLNNLTV (SEQ ID NO:546), TNIDSTP (SEQ ID NO:547), TNIDSTP (SEQ ID NO:548), LRILANK (SEQ ID NO:549), LLTPTLN (SEQ ID NO:550), ALPTHSN (SEQ ID NO:551), TNIDSTP (SEQ ID NO:552), LCRRFEN (SEQ ID NO:553), TNIDSTP (SEQ ID NO:554), TNIDSTP (SEQ ID NO:555), HLQTTET (SEQ ID NO:556), PLGLCPP (SEQ ID NO:557), GYFIPSI (SEQ ID NO:558), TKIDSTP (SEQ ID NO:559), HLQTTET (SEQ ID NO:560), WNIDSTP (SEQ ID NO:561), TWIDWTP (SEQ ID NO:562), RTQPYPL (SEQ ID NO:670) and TWIDSTP (SEQ ID NO:671).

10. The polynucleotide of claim 7 , wherein said binding peptide is a TGFβ binding peptide selected from the group consisting of: CLCPENINVLPCN (PEN3; SEQ ID NO:436), CICKHNVDWLCF (MMO21W; SEQ ID NO:437), CICWTQHIHNCF (WTQ; SEQ ID NO:438), CVTTDWIEC (SEQ ID NO:563), CYYSQFHQC (SEQ ID NO:564), CPTLWTHMC (SEQ ID NO:565), QSACIVYYVGRKPKVECASSD (SEQ ID NO:566), QSACILYYIGKTPKIECASSD (SEQ ID NO:567), QSACILYYVGRTPKVECASSD (SEQ ID NO:568), KHNVRLL (SEQ ID NO:570), NDTPSYF (SEQ ID NO:571), AKLYAGS (SEQ ID NO:572), RGPAHSL (SEQ ID NO:573), NSLAERR (SEQ ID NO:574), HPLASPH (SEQ ID NO:575), QPWNKLK (SEQ ID NO:576), PTKPAQQ (SEQ ID NO:578), PSLNRPQ (SEQ ID NO:579), HHARQEW (SEQ ID NO:580), RHHTPGP (SEQ ID NO:581), ASAINPH (SEQ ID NO:582), CHGYDRAPC (SEQ ID NO:644), CFAPADQAC (SEQ ID NO:645), CIPSRFITC (SEQ ID NO:646), CHGHTKLAC (SEQ ID NO:647), CNGKSKLAC (SEQ ID NO:648), PENINVLP (SEQ ID NO:672), KHNVDWL (SEQ ID NO:673) and WTQHIHNC (SEQ ID NO:674).

11. The polynucleotide of claim 7 , wherein said binding peptide is a TNFα binding peptide selected from the group consisting of: RYWQDIP (T1; SEQ ID NO:474), APEPILA (T2; SEQ ID NO:475), DMIMVSI (T3; SEQ ID NO:476), WTPKPTQ (SEQ ID NO:583), ATFPNQS (SEQ ID NO:584), ASTVGGL (SEQ ID NO:585), TMLPYRP (SEQ ID NO:586), AWHSPSV (SEQ ID NO:587), TQSFSS (SEQ ID NO:588), THKNTLR (SEQ ID NO:589), GQTHFHV (SEQ ID NO:590), LPILTQT (SEQ ID NO:591), SILPVSH (SEQ ID NO:592), SQPIPI (SEQ ID NO:593), and QPLRKLP (SEQ ID NO:594).

12. The polynucleotide of claim 1 , wherein the backbone of the unmodified precursor variant BBPI comprises a BBPI scaffold selected from the group consisting of: BBI (SEQ ID NO:13), BBIt (SEQ ID NO:185), BBI-AV (SEQ ID NO:186), BBIt-AV (SEQ ID NO:187), BBIt-VEGK (SEQ ID NO:640), BBIt-VEGT (SEQ ID NO:641), BBIt-VEGKD (SEQ ID NO:642), BBdb (SEQ ID NO:449), BBsb3 (SEQ ID NO:450), BBtc (SEQ ID NO:451), BBdb-AV (SEQ ID NO:452), BBsb3-AV (SEQ ID NO:453) and BBtc-AV (SEQ ID NO:454).

13. The polynucleotide of claim 1 , wherein the backbone of the unmodified precursor variant BBPI is substituted at a combination of two amino acid positions equivalent to positions 50 and 52 of SEQ ID NO:187.

14. The polynucleotide of claim 1 , wherein the backbone of the unmodified precursor variant BBPI is substituted at a combination of three amino acid positions chosen from amino acid positions equivalent to positions 25, 29, 40, 50 and 52 of SEQ ID NO:187.

15. The polynucleotide of claim 1 , wherein the backbone of the unmodified precursor variant BBPI is substituted at a combination of four amino acid positions chosen from amino acid positions equivalent to positions 13, 25, 29, 40, 50 and 52 of SEQ ID NO:187.

16. The polynucleotide of claim 1 , wherein the backbone of the unmodified precursor variant BBPI is substituted at a combination of five amino acid positions selected from amino acid positions equivalent to positions 13, 25, 29, 40, 50, and 52 of SEQ ID NO:187.

17. The polynucleotide of claim 1 , wherein the backbone of the unmodified precursor variant BBPI is substituted at a combination of six amino acid positions chosen from amino acid positions equivalent to positions 1, 4, 5, 11, 13, 25, 27, 29, 31, 38, 40, 50, 52 and 65 of SEQ ID NO:187.

18. The polynucleotide of claim 1 , wherein the backbone of the unmodified precursor variant BBPI is substituted at a combination of seven amino acid positions equivalent to positions 13, 25, 29, 31, 40, 50 and 52 of SEQ ID NO:187.

19. The polynucleotide of claim 1 , wherein the backbone of the unmodified precursor variant BBPI is substituted at a combination of eight amino acid positions equivalent to positions 13, 25, 27, 29, 31, 40, 50 and 52 of SEQ ID NO:187.

20. The polynucleotide of claim 1 , wherein said modified variant BBPI comprises a combination of amino acid substitutions selected from the group consisting of: 25L-50T-52A, 29P-50T-52A, 40K-50T-52A, 13I-25L-50T-52A, 13I-29P-50T-52A, 13I-40K-50T-52A, 25L-29P-50T-52A, 25L-40K-50T-52A, 29P-40K-50T-52A, 13I-25K-29P-52K, 13I-25L-29P-50T-52A, 13I-25L-40K-50T-52A, 13I-29P-40K-50T-52A, 25L-29P-40K-50T-52T, 13L-29P-40K-50T-52A, 13I-29P-40K-50K-52A, 13L-29P-40K-50T-52T, 13I-25L-29P-40K-50T-52A, 1C-13I-29P-40K-50T-52A, 4V-13I-29P-40K-50T-52A, 5P-13I-29P-40K-50T-52A, 11G-13I-29P-40K-50T-52A, 13I-25R-29P-40K-50T-52A, 13I-27R-29P-40K-50T-52A, 13I-29P-31A-40K-50T-52A, 13I-29P-31R-40K-50T-52A, 13I-29P-38N-40K-50T-52A, 13I-29P-40K-50T-52A-65E, 13I-25R-27A-29P-31A-50K-52T, 13L-25R-29P-31A-40K-50T-52A, 13L-25R-29P-31R-40K-50T-52A, 13I-25R-27A-29P-31A-40H-50K-52T, 13I-25K-27A-29R-31E-40K-50Q-52Q, 13I-25K-27R-29E-31A-40H-50R-52K, 13I-25K-27A-29R-31A-40H-50R-52L, and 13I-25K-27Q-29P-31E-40H-50R-52Q.

21. The polynucleotide of claim 1 , wherein said modified variant BBPI comprises SEQ ID NO: 432, 434, 443, 445, 447, 491, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639 or 643.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 28, 2011
From: AMIN, NEELAM S.; COLLIER, KATHERINE D.; ESTABROOK, MELODIE; ESTELL, DAVID A.; FOX, BRYAN P.; POWER, SCOTT D.; SCHMIDT, BRIAN F.; VOGTENTANZ, GUDRUN
To: DANISCO US INC.
Reel/Frame 027143/0427 →
Continuity (3)
Continuation 12779793 · May 13, 2010
Division 12252062 · Oct 15, 2008
Related Publication 20110256608A1 · Oct 20, 2011