Synthetic genes for plant gums and other hydroxyproline-rich glycoproteins

Abstract

A new approach in the field of plant gums is described which presents a new solution to the production of hydroxyproline(Hyp)-rich glycoproteins (HRGPs), repetitive proline-rich proteins (RPRPs) and arabinogalactan-proteins (AGPs). The expression of synthetic genes designed from repetitive peptide sequences of such glycoproteins, including the peptide sequences of gum arabic glycoprotein (GAGP), is taught in host cells, including plant host cells.

I claim:  
     
         1 . An isolated polynucleotide sequence encoding at least a portion of an amino acid sequence selected from (a) A-Hyp-B-C-D-E-F-Hyp-G-H-I-Hyp-J-Hyp-Hyp-K-L-Pro-M (SEQ ID NO:136), wherein A is selected from Ser, Thr, and Ala; B is selected from Hyp, Pro, Leu, and Ile; C is selected from Pro and Hyp; D is selected from Hyp, Pro, Ser, Thr, and Ala; E is selected from Leu and Ile; F is selected from Ser, Thr, and Ala; G is selected from Ser, Leu, Hyp, Thr, Ala, and Ile; H is selected from Hyp, Pro, Leu, and Ile; I is selected from Thr, Ala, and Ser; J is selected from Thr, Ser, and Ala; K is selected from Thr, Leu, Hyp, Ser, Ala, and Ile; L is selected from Gly, Leu, Ala, and Ile; and M is selected from His and Pro; and wherein said portion is greater than twelve contiguous amino acids of said amino acid sequence, (b) a polypeptide comprising a first motif selected from (Xaa-Hyp) x  (SEQ ID NO:182) and Xaa-Hyp-Xaa-Xaa-Hyp-Xaa (SEQ ID NO: 183), wherein x is from 2 to 1000, (c) a polypeptide comprising a second motif selected from Xaa-Hyp-Hyp n  (SEQ ID NO:209) and Xaa-Pro-Hypn (SEQ ID NO:210), wherein n is from 1 to 500, and (d) a polypeptide comprising said first motif and said second motif, wherein Xaa is any amino acid other than hydroxyproline.  
     
     
         2 . A recombinant expression vector comprising a polynucleotide sequence encoding a portion of an amino acid sequence selected from (a) A-Hyp-B-C-D-E-F-Hyp-G-H-I-Hyp-J-Hyp-Hyp-K-L-Pro-M (SEQ ID NO:136), wherein A is selected from Ser, Thr, and Ala; B is selected from Hyp, Pro, Leu, and Ile; C is selected from Pro and Hyp; D is selected from Hyp, Pro, Ser, Thr, and Ala; E is selected from Leu and Ile; F is selected from Ser, Thr, and Ala; G is selected from Ser, Leu, Hyp, Thr, Ala, and Ile; H is selected from Hyp, Pro, Leu, and Ile; I is selected from Thr, Ala, and Ser; J is selected from Thr, Ser, and Ala; K is selected from Thr, Leu, Hyp, Ser, Ala, and Ile; L is selected from Gly, Leu, Ala, and Ile; and M is selected from His and Pro; and wherein said portion is greater than twelve contiguous amino acids of said amino acid sequence, (b) a polypeptide comprising a first motif selected from (Xaa-Hyp) x  (SEQ ID NO:182) and Xaa-Hyp-Xaa-Xaa-Hyp-Xaa (SEQ ID NO:183), wherein x is from 2 to 1000, (c) a polypeptide comprising a second motif selected from Xaa-Hyp-Hyp n  (SEQ ID NO:209) and Xaa-Pro-Hyp n  (SEQ ID NO:210), wherein n is from 1 to 500, and (d) a polypeptide comprising said first motif and said second motif, wherein Xaa is any amino acid other than hydroxyproline.  
     
     
         3 . The expression vector of  claim 2 , further comprising a promoter operably linked to said polynucleotide sequence.  
     
     
         4 . The expression vector of  claim 3 , wherein said promoter is a viral promoter.  
     
     
         5 . The expression vector of  claim 4 , wherein said viral promoter is selected from the group consisting of the 35S and 19S RNA promoters of cauliflower mosaic virus.  
     
     
         6 . The expression vector of  claim 3 , further comprising a signal sequence selected from extensin signal sequence (SEQ ID NO:14), and tomato arabinogalactan-protein signal sequence (SEQ ID NO:215).  
     
     
         7 . The expression vector of  claim 6 , further comprising a reporter gene.  
     
     
         8 . The expression vector of  claim 7 , wherein said reporter gene is the green fluorescence protein gene.  
     
     
         9 . The expression vector of  claim 2 , wherein said vector is contained within a host cell.  
     
     
         10 . The expression vector of  claim 9 , wherein said host cell is a plant cell.  
     
     
         11 . The expression vector of  claim 10 , wherein said plant cell expresses a glycoprotein comprising said portion.  
     
     
         12 . A method for producing at least a portion of a glycoprotein, comprising: 
 a) providing: 
 i) a recombinant expression vector comprising a polynucleotide sequence encoding at least a portion of an amino acid sequence selected from (a) A-Hyp-B-C-D-E-F-Hyp-G-H-I-Hyp-J-Hyp-Hyp-K-L-Pro-M (SEQ ID NO:136), wherein A is selected from Ser, Thr, and Ala; B is selected from Hyp, Pro, Leu, and Ile; C is selected from Pro and Hyp; D is selected from Hyp, Pro, Ser, Thr, and Ala; E is selected from Leu and Ile; F is selected from Ser, Thr, and Ala; G is selected from Ser, Leu, Hyp, Thr, Ala, and Ile; H is selected from Hyp, Pro, Leu, and Ile; I is selected from Thr, Ala, and Ser; J is selected from Thr, Ser, and Ala; K is selected from Thr, Leu, Hyp, Ser, Ala, and Ile; L is selected from Gly, Leu, Ala, and Ile; and M is selected from His and Pro; and wherein said portion is greater than twelve contiguous amino acids of said amino acid sequence, (b) a polypeptide comprising a first motif selected from (Xaa-Hyp) x  (SEQ ID NO:182) and Xaa-Hyp-Xaa-Xaa-Hyp-Xaa (SEQ ID NO:183), wherein x is from 2 to 1000, (c) a polypeptide comprising a second motif selected from Xaa-Hyp-Hyp n  (SEQ ID NO:209) and Xaa-Pro-Hyp n  (SEQ ID NO:210), wherein n is from 1 to 500, and (d) a polypeptide comprising said first motif and said second motif, wherein Xaa is any amino acid other than hydroxyproline; and  
 ii) a host cell; and  
   b) introducing said vector into said host cell under conditions such that said portion is expressed.    
     
     
         13 . The method of  claim 12 , wherein said host cell is growing in culture.  
     
     
         14 . The method of  claim 13 , further comprising the step of c) recovering said portion from the host cell culture.  
     
     
         15 . The method of  claim 12 , wherein said host cell is a plant cell.  
     
     
         16 . The method of  claim 15 , wherein said plant cell is derived from a plant selected from the family Leguminoseae.