Expression of biologically active polypeptides in duckweed

Abstract

Methods, nucleic acid sequences, and transformed duckweed plant or duckweed nodule cultures for the expression and the secretion of biologically active polypeptides from genetically engineered duckweed are provided. Expression of recombinant polypeptides in duckweed is improved by modifying the nucleotide sequence of the expression cassette encoding the polypeptide for improved expression in duckweed. Recovery of biologically active polypeptides from duckweed is improved by linking the biologically active polypeptide to a signal peptide that directs the secretion of the polypeptide into the culture medium.

1 . A duckweed plant culture or a duckweed nodule culture, wherein said duckweed plant culture or said duckweed nodule culture is stably transformed with a nucleic acid molecule comprising a coding sequence for α-2b-interferon and an operably linked coding sequence for a signal peptide capable of directing secretion of the α-2b-interferon. 
     
     
         2 . The duckweed plant culture or duckweed nodule culture of  claim 1 , wherein said nucleic acid molecule has at least one attribute selected from the group consisting of:
 (a) duckweed-preferred codons in the coding sequence for said α-2b-interferon;   (b) duckweed-preferred codons in the coding sequence for said signal peptide;   (c) a translation initiation codon that is flanked by a plant-preferred translation initiation context nucleotide sequence, wherein said plant-preferred translation initiation context nucleotide sequence consists of the nucleotide sequence “ACC” or “ACA”, wherein said context is positioned immediately adjacent to the 5′ end of the translation initiation codon; and   (d) an operably linked nucleotide sequence comprising a plant intron that is inserted upstream of the coding sequence; and   (e) an operably linked 5′ leader sequence.   
     
     
         3 . The duckweed plant culture or duckweed nodule culture according to  claim 2 , wherein said duckweed-preferred codons are  Lemna gibba -preferred codons or  Lemna minor -preferred codons. 
     
     
         4 . The duckweed plant culture or duckweed nodule culture according to  claim 3 , wherein at least one coding sequence selected from the coding sequence for said polypeptide and the coding sequence for said signal peptide comprises between 70-100%  Lemna gibba -preferred codons or  Lemna minor -preferred codons. 
     
     
         5 . The duckweed plant culture or duckweed nodule culture according to  claim 4 , wherein said nucleic acid molecule has a 5′ leader sequence operably linked to the coding sequence for said α-2b interferon. 
     
     
         6 . The duckweed plant culture or duckweed nodule culture according to  claim 4 , wherein said nucleic acid molecule further comprises an operably linked nucleotide sequence comprising a plant intron that is inserted upstream of the coding sequence. 
     
     
         7 . The duckweed plant culture or duckweed nodule culture according to  claim 5 , wherein said nucleotide sequence further comprises an operably linked nucleotide sequence comprising a plant intron that is inserted upstream of the coding sequence. 
     
     
         8 . The duckweed plant culture or duckweed nodule culture according to  claim 7 , wherein said operably linked nucleotide sequence comprising said plant intron is the sequence set forth in SEQ ID NO:1. 
     
     
         9 . A duckweed plant culture or a duckweed nodule culture, wherein said duckweed plant culture or said duckweed nodule culture is stably transformed with a nucleic acid molecule comprising a nucleotide sequence encoding biologically active α-2b-interferon wherein said nucleotide sequence has been modified for enhanced expression in duckweed. 
     
     
         10 . The duckweed plant culture or duckweed nodule culture of  claim 9 , wherein said nucleotide sequence that has been modified for enhanced expression in duckweed has at least one attribute selected from the group consisting of:
 (a) duckweed-preferred codons in the coding sequence for said biologically active α-2b-interferon;   (b) a translation initiation codon that is flanked by a plant-preferred translation initiation context nucleotide sequence, wherein said plant-preferred translation initiation context nucleotide sequence consists of the nucleotide sequence “ACC” or “ACA”, wherein said context is positioned immediately adjacent to the 5′ end of the translation initiation codon; and   (c) an operably linked nucleotide sequence comprising a plant intron that is inserted upstream of the coding sequence; and   (d) an operably linked 5′ leader sequence.   
     
     
         11 . The duckweed plant culture or duckweed nodule culture of  claim 10 , wherein said duckweed-preferred codons are  Lemna gibba -preferred codons or  Lemna minor -preferred codons. 
     
     
         12 . The duckweed plant culture or duckweed nodule culture of  claim 11 , wherein the coding sequence comprises between 70% and 100%  Lemna gibba -preferred codons or  Lemna minor -preferred codons. 
     
     
         13 . The duckweed plant culture or duckweed nodule culture according to  claim 12 , wherein said nucleic acid molecule has a 5′ leader sequence operably linked to the coding sequence for said α-2b interferon. 
     
     
         14 . The duckweed plant culture or duckweed nodule culture according to  claim 11 , wherein said nucleic acid molecule further comprises an operably linked nucleotide sequence comprising a plant intron that is inserted upstream of the coding sequence. 
     
     
         15 . The duckweed plant culture or duckweed nodule culture according to  claim 13 , wherein said nucleic acid molecule further comprises an operably linked plant intron that is inserted upstream of the coding sequence. 
     
     
         16 . The duckweed plant culture or duckweed nodule culture according to  claim 15 , wherein said plant intron is the sequence set forth in SEQ ID NO:1. 
     
     
         17 . The duckweed plant culture or duckweed nodule culture according to  claim 1 , wherein said α-2b-interferon is a human α-2b-interferon. 
     
     
         18 . The duckweed plant culture or duckweed nodule culture according to  claim 17 , wherein said human α-2b-interferon has an amino acid sequence selected from the group consisting of the amino acid sequence set forth in SEQ ID NO:4 and the amino acid sequence set forth in SEQ ID NO:5. 
     
     
         19 . The duckweed plant culture or duckweed nodule culture according to  claim 1 , wherein the α-2b-interferon is biologically active and has at least 80% sequence identity with an amino acid sequence selected from the group consisting of the amino acid sequence set forth in SEQ ID NO:4 and the amino acid sequence set forth in SEQ ID NO:5. 
     
     
         20 . The duckweed plant culture or duckweed nodule culture according to  claim 19 , wherein the α-2b-interferon comprises a polypeptide that is a terminal truncation of the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5. 
     
     
         21 . The duckweed plant culture or duckweed nodule culture according to  claim 20 , wherein said terminal truncation is a C-terminal truncation. 
     
     
         22 . The duckweed plant culture or duckweed nodule culture according to  claim 1 , wherein said signal peptide is selected from the group consisting of:
 (a) the human α-2b-interferon signal peptide;   (b) the  Arabidopsis thaliana  chitinase signal peptide;   (c) the rice α-amylase signal peptide;   (d) the modified rice α-amylase peptide;   (e) a duckweed signal peptide; and   (f) a signal peptide native to the α-2b interferon.   
     
     
         23 . The duckweed plant culture or duckweed nodule culture according to  claim 22 , wherein the signal peptide is the rice α-amylase signal peptide having the sequence set forth in SEQ ID NO:3. 
     
     
         24 . The duckweed plant culture or duckweed nodule culture according to  claim 1 , wherein said duckweed plant culture or duckweed nodule culture is selected from the group consisting of the genus  Spirodela , genus  Wolffia , genus  Wolfiella , and genus  Lemna.    
     
     
         25 . The duckweed plant culture or duckweed nodule culture according to  claim 24 , wherein said duckweed plant culture or duckweed nodule culture is selected from the group consisting of  Lemna minor, Lemna miniscula, Lemna aequinoctialis , and  Lemna gibba.    
     
     
         26 . The duckweed plant culture or duckweed nodule culture according to  claim 9 , wherein said duckweed plant culture or duckweed nodule culture is selected from the group consisting of the genus  Spirodela , genus  Wolffia , genus  Wolfiella , and genus  Lemna.    
     
     
         27 . The duckweed plant culture or duckweed nodule culture according to  claim 26 , wherein said duckweed plant culture or duckweed nodule culture is selected from the group consisting of  Lemna minor, Lemna miniscula, Lemna aequinoctialis , and  Lemna gibba.    
     
     
         28 . A nucleic acid molecule comprising the α-2b-interferon-encoding nucleotide sequence set forth in SEQ ID NO:2 operably linked to the signal peptide-encoding nucleotide sequence given in SEQ ID NO:3. 
     
     
         29 . The nucleic acid molecule of  claim 28 , wherein said nucleic acid molecule further comprises the plant intron nucleotide sequence given in SEQ ID NO:1, wherein the plant intron nucleotide sequence, the signal peptide-encoding nucleotide sequence and the α-2b-interferon-encoding nucleotide sequence are operably linked. 
     
     
         30 . A duckweed plant culture or duckweed nodule culture, stably transformed to express a biologically active recombinant polypeptide from a nucleotide sequence comprising a coding sequence for said recombinant polypeptide and an operably linked coding sequence for a signal peptide that directs secretion of the recombinant polypeptide; wherein said signal peptide sequence is selected from the group consisting of:
 (a) the human α-2b-interferon signal peptide;   (b) the  Arabidopsis thaliana  chitinase signal peptide;   (c) the rice α-amylase signal peptide; and   (d) the modified rice α-amylase peptide set forth in SEQ ID NO:7.   
     
     
         31 . The duckweed plant culture or duckweed nodule culture according to  claim 30 , wherein said recombinant polypeptide is an α-2b-interferon having at least 80% sequence identity with an amino acid sequence selected from the group consisting of the amino acid sequence set forth in SEQ ID NO:4 and the amino acid sequence set forth in SEQ ID NO:5. 
     
     
         32 . A method of producing biologically active recombinant polypeptide in a duckweed plant culture or a duckweed nodule culture, comprising the steps of:
 (a) culturing within a duckweed culture medium a duckweed plant culture or a duckweed nodule culture, wherein said duckweed plant culture or said duckweed nodule culture is stably transformed to express said biologically active recombinant polypeptide, and wherein said biologically active recombinant polypeptide is expressed from a nucleotide sequence comprising a coding sequence for said recombinant polypeptide and an operably linked coding sequence for a signal peptide that directs secretion of the recombinant polypeptide; and   (b) collecting said recombinant polypeptide from the duckweed culture medium;   
       wherein said signal peptide is selected from the group consisting of:
 (i) the human α-2b-interferon signal peptide; 
 (ii) the  Arabidopsis thaliana  chitinase signal peptide; 
 (iii) the rice α-amylase signal peptide; and 
 (iv) the modified rice α-amylase peptide set forth in SEQ ID NO:7. 
 
     
     
         33 . The method of  claim 32 , wherein said recombinant polypeptide is an α-2b-interferon having at least 80% sequence identity with an amino acid sequence selected from the group consisting of the amino acid sequence set forth in SEQ ID NO:4 and the amino acid sequence set forth in SEQ ID NO:5.