US2005287637A1PendingUtilityA1

Engineering intracellular sialylation pathways

58
Individually held — no corporate assignee on recordPriority: Mar 2, 1999Filed: May 6, 2005Published: Dec 29, 2005
Est. expiryMar 2, 2019(expired)· nominal 20-yr term from priority
Inventors:Michael J. BetenbaughShawn LawrenceYuan-Chuan LeeTimothy A. Coleman
C12N 15/52C12P 21/005
58
PatentIndex Score
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Claims

Abstract

Methods for manipulating carbohydrate processing pathways in cells of interest are provided. Methods are directed at manipulating multiple pathways involved with the sialylation reaction by using recombinant DNA technology and substrate feeding approaches to enable the production of sialylated glycoproteins in cells of interest. These carbohydrate engineering efforts encompass the implementation of new carbohydrate bioassays, the examination of a selection of insect cell lines and the use of bioinformatics to identify gene sequences for critical processing enzymes. The compositions comprise cells of interest producing sialylated glycoproteins. The methods and compositions are useful for heterologous expression of glycoproteins.

Claims

exact text as granted — not AI-modified
1 - 25 . (canceled)  
     
     
         26 . A method for manipulating glycoprotein production in a cell, said method comprising enhancing expression of at least one enzyme selected from the group consisting of: 
 a) GlcNAc-2 epimerase;    b) an enzyme catalyzing conversion of UDP-GlcNAc to ManNAc;    c) sialic acid synthetase;    d) aldolase;    e) CMP-SA synthetase;    f) CMP-SA transporter; and    wherein expression of each enzyme expressed is enhanced to above endogenous levels.    
     
     
         27 . The method of  claim 26 , wherein expression of enzyme (a) is enhanced.  
     
     
         28 . The method of  claim 27 , wherein said enzyme is human.  
     
     
         29 . The method of  claim 26 , wherein expression of enzyme (b) is enhanced.  
     
     
         30 . The method of  claim 29 , wherein said enzyme is human.  
     
     
         31 . The method of  claim 26 , wherein expression of enzyme (c) is enhanced.  
     
     
         32 . The method of  claim 31 , wherein said enzyme has the sequence of SEQ ID NO:6.  
     
     
         33 . The method of  claim 26 , wherein expression of enzyme (d) is enhanced.  
     
     
         34 . The method of  claim 33 , wherein said enzyme has the sequence of SEQ ID NO: 2.  
     
     
         35 . The method of  claim 26 , wherein expression of enzyme (e) is enhanced.  
     
     
         36 . (canceled)  
     
     
         37 . The method of  claim 26 , wherein expression of enzyme (f) is enhanced.  
     
     
         38 . The method of  claim 37 , wherein said enzyme is human.  
     
     
         39 . (canceled)  
     
     
         40 . The method of  claim 26 , further comprising suppressing activity of endogenous N-acetylglucoaminidase.  
     
     
         41 . A method for producing sialylated glycoproteins, said method comprising expressing a heterologous protein in a cell manipulated according to the method of  claim 26 .  
     
     
         42 . The method of  claim 41 , wherein said heterologous protein is mammalian.  
     
     
         43 . The method of  claim 42 , wherein said mammalian protein is selected from the group consisting of plasminogen, transferrin, Na+, K+-ATPase, and thyrotropin.  
     
     
         44 - 46 . (canceled)  
     
     
         47 . The method of  claim 26 , wherein expression of both enzyme c) and enzyme (e) is enhanced.  
     
     
         48 . The method of  claim 26  wherein said cell is selected from the group consisting of yeast, insect, fungal, plant, mammalian and bacterial cells.  
     
     
         49 . The method of  claim 41  wherein said cell is selected from the group consisting of yeast, insect, fungal, plant, mammalian and bacterial cells.  
     
     
         50 . The method of  claim 26  wherein said method is used to produce a sialylated glycoprotein in said cell.  
     
     
         51 . The method of  claim 50 , wherein said sialylated glycoprotein is a heterologous protein.  
     
     
         52 . The method of  claim 35 , wherein said enzyme has the sequence of SEQ ID NO: 4.  
     
     
         53 . The method of  claim 26 , wherein expression of enzymes b), c), and e) is enhanced.  
     
     
         54 . The method of  claim 26 , wherein expression of enzymes b), c), e) and f) is enhanced.

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