US2017204381A1PendingUtilityA1

Pmst1 mutants for chemoenzymatic synthesis of sialyl lewis x compounds

44
Assignee: UNIV CALIFORNIAPriority: Aug 5, 2011Filed: Jan 8, 2016Published: Jul 20, 2017
Est. expiryAug 5, 2031(~5 yrs left)· nominal 20-yr term from priority
Inventors:Xi ChenGo SugiartoKam-Yin Lau
C12N 9/1081C12N 9/1077C12P 19/04C12Y 204/99C12P 19/18
44
PatentIndex Score
0
Cited by
0
References
0
Claims

Abstract

The present invention provides mutants of PmST1 for the preparation of sialyl-Lewis x oligosaccharides, and other sialosides with decreased sialidase activity.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
         1 . An isolated glycosyltransferase, wherein
 the amino acid corresponding to position 120 of SEQ ID NO:1 is any amino acid other than M,   the amino acid corresponding to position 247 of SEQ ID NO:1 is any amino acid other than E, or   the amino acid corresponding to position 289 of SEQ ID NO:1 is any amino acid other than R,   wherein the glycosyltransferase has decreased α2-3 sialidase or donor substrate hydrolysis activity compared to a control glycosyltransferase, wherein the amino acid of the glycosyltransferase corresponding to position 120 of SEQ ID NO:1 is M, the amino acid corresponding to position 247 of SEQ ID NO:1 is E, and the amino acid corresponding to position 289 of SEQ ID NO:1 is R,   and wherein the glycosyltransferase is a member of the glycosyltransferase family 80 (GT80).   
     
     
         2 . The isolated glycosyltransferase of  claim 1 , wherein the isolated glycosyltransferase has decreased α2-3 sialidase activity, and
 the amino acid of the glycosyltransferase corresponding to position 247 of SEQ ID NO:1 is any amino acid other than E, or 
 the amino acid of the glycosyltransferase corresponding to position 289 of SEQ ID NO:1 is any amino acid other than R. 
 
     
     
         3 . The isolated glycosyltransferase of any of  claims 1 - 2 , wherein the ratio of α2-3 sialidase activity for the control glycosyltransferase to the α2-3 sialidase activity of the isolated glycosyltransferase is at least 5:1. 
     
     
         4 . The isolated glycosyltransferase of  claim 3 , wherein the ratio is at least 10:1. 
     
     
         5 . The isolated glycosyltransferase of  claim 3 , wherein the ratio is at least 100:1. 
     
     
         6 . The isolated glycosyltransferase of  claim 3 , wherein the ratio is at least 1000:1. 
     
     
         7 . The isolated glycosyltransferase of any of  claims 1 - 6 , wherein the isolated glycosyltransferase comprises:
 the amino acid corresponding to position 247 of SEQ ID NO:1 is any amino acid other than E, and   the amino acid corresponding to position 289 of SEQ ID NO:1 is any amino acid other than R.   
     
     
         8 . The isolated glycosyltransferase of any of  claims 1 - 7 , wherein the isolated glycosyltransferase has decreased donor substrate hydrolysis activity, and wherein the amino acid corresponding to position 120 of SEQ ID NO:1 is any amino acid other than M. 
     
     
         9 . The isolated glycosyltransferase of  claim 8 , wherein the ratio of donor substrate hydrolysis activity for the control α2-3 sialidase to the donor substrate hydrolysis activity of the isolated glycosyltransferase is at least 2:1. 
     
     
         10 . The isolated glycosyltransferase of any of  claims 1 - 9 , wherein the amino acid corresponding to position 120 of SEQ ID NO:1 is any amino acid selected from the group consisting of V, I, L, F, W, P, S, T, A, G, C, Y, N, Q, D, E, K, R, and H. 
     
     
         11 . The isolated glycosyltransferase of any of  claims 1 - 10 , wherein the amino acid corresponding to position 247 of SEQ ID NO:1 is any amino acid selected from the group consisting of V, I, L, M, F, W, P, S, T, A, G, C, Y, N, Q, D, K, R, and H. 
     
     
         12 . The isolated glycosyltransferase of any of  claims 1 - 11 , wherein the amino acid corresponding to position 289 of SEQ ID NO:1 is any amino acid selected from the group consisting of V, I, L, M, F, W, P, S, T, A, G, C, Y, N, Q, D, E, K, and H. 
     
     
         13 . The isolated glycosyltransferase of any of  claims 1 - 12 , wherein
 the amino acid corresponding to position 120 of SEQ ID NO:1 is D, E, H, K or R,   the amino acid corresponding to position 247 of SEQ ID NO:1 is F, Y or W, or   the amino acid corresponding to position 289 of SEQ ID NO:1 is Y, F or W.   
     
     
         14 . The isolated glycosyltransferase of any of  claims 1 - 13 , wherein
 the amino acid corresponding to position 120 of SEQ ID NO:1 is D or H,   the amino acid corresponding to position 247 of SEQ ID NO:1 is F, or   the amino acid corresponding to position 289 of SEQ ID NO:1 is Y.   
     
     
         15 . The isolated glycosyltransferase of any of  claims 1 - 14 , wherein the glycosyltransferase is an α2-3 sialyltransferase. 
     
     
         16 . The isolated glycosyltransferase of  claim 15 , comprising a motif in the sialyltransferase domain comprising at least one member selected from the group consisting of sialyltransferase motif A (YDDGS) and sialyltransferase motif B (KGH). 
     
     
         17 . The isolated glycosyltransferase of any of  claims 1 - 16 , wherein the control glycosyltransferase is SEQ ID NO:1. 
     
     
         18 . The isolated glycosyltransferase of  claim 17 , wherein the glycosyltransferase comprises a polypeptide sequence having at least 80% sequence identity to SEQ ID NO:1. 
     
     
         19 . The isolated glycosyltransferase of  claim 1 , wherein the isolated glycosyltransferase comprises a polypeptide sequence selected from the group consisting of SEQ ID NO: 3 (M120D), SEQ ID NO: 5 (M120H), SEQ ID NO: 7 (E247F), SEQ ID NO: 9 (R289Y) and SEQ ID NO: 11 (E247F/R289Y). 
     
     
         20 . A recombinant nucleic acid encoding an isolated glycosyltransferase of any of  claims 1 - 19 . 
     
     
         21 . A cell comprising an recombinant nucleic acid of  claim 20 . 
     
     
         22 . The cell of  claim 21 , wherein the cell is selected from the group consisting of bacteria, yeast, insect, mammalian and plant cells. 
     
     
         23 . A method of preparing an oligosaccharide, the method comprising:
 a) forming a reaction mixture comprising an acceptor sugar, a donor substrate comprising a sugar moiety and a nucleotide, and the glycosyltransferase of any of  claims 1 - 19 , under conditions sufficient to transfer the sugar moiety from the donor substrate to the acceptor sugar, thereby forming the oligosaccharide.   
     
     
         24 . The method of  claim 23 , wherein the glycosyltransferase comprises a polypeptide sequence selected from the group consisting of SEQ ID NO: 3 (M120D), SEQ ID NO: 5 (M120H), SEQ ID NO: 7 (E247F), SEQ ID NO: 9 (R289Y) and SEQ ID NO: 11 (E247F/R289Y). 
     
     
         25 . The method of  claim 23 , wherein the glycosyltransferase comprises a polypeptide sequence selected from the group consisting of SEQ ID NO: 3 (M120D) and SEQ ID NO: 5 (M120H). 
     
     
         26 . The method of  claim 23 , wherein the isolated glycosyltransferase comprises a polypeptide sequence selected from the group consisting of SEQ ID NO: 7 (E247F), SEQ ID NO: 9 (R289Y) and SEQ ID NO: 11 (E247F/R289Y). 
     
     
         27 . The method of  claim 23 , wherein the donor substrate comprises a cytidine 5′-monophosphate(CMP)-sialic acid. 
     
     
         28 . The method of  claim 27 , wherein the CMP-sialic acid comprises cytidine 5′-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac) or a CMP-Neu5Ac analog. 
     
     
         29 . The method of  claim 28 , further comprising:
 b) forming a reaction mixture comprising a CMP-sialic acid synthetase, cytidine triphosphate, and N-acetylneuraminic acid (Neu5Ac) or a Neu5Ac analog, under conditions suitable to form the CMP-Neu5Ac or CMP-Neu5Ac analog.   
     
     
         30 . The method of  claim 29 , wherein steps a) and b) are performed in one pot. 
     
     
         31 . The method of  claim 29 , further comprising:
 c) forming a reaction mixture comprising a sialic acid aldolase, pyruvic acid or derivatives thereof, and N-acetylmannosamine or derivatives thereof, under conditions suitable to form the Neu5Ac or Neu5Ac analog.   
     
     
         32 . The method of  claim 31 , wherein steps a), b), and c) are performed in one pot. 
     
     
         33 . The method of any of  claims 23 - 32 , wherein the oligosaccharide is an ci2-3-linked sialyloligosaccharide. 
     
     
         34 . The method of  claim 23 , wherein the oligosaccharide is a fucosylated oligosaccharide.

Join the waitlist — get patent alerts

Track US2017204381A1 — get alerts on status changes and closely related new filings.

We store only your email — no account needed. See our privacy policy.