Claims for Patent: 6,815,184

Summary for Patent: 6,815,184

Title:	Expression of biologically active polypeptide 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.
Inventor(s):	Stomp; Anne-Marie (Moncure, NC), Dickey; Lynn (Cary, NC), Gasdaska; John (Carrboro, NC)
Assignee:	Biolex, Inc. (Pittsboro, NC)
Application Number:	09/915,873
Patent Claims:	1. A method of producing biologically active .alpha.-2b-interferon 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 .alpha.-2b-interferon, and wherein said biologically active .alpha.-2b-interferon is expressed from a nucleotide sequence comprising a coding sequence for the polypeptide and an operably linked coding sequence for a signal peptide that directs secretion of the .alpha.-2b-interferon into the culture medium; and (b) collecting said biologically active .alpha.-2b interferon from the duckweed culture medium. 2. The method of claim 1, wherein said biologically active .alpha.-2b-interferon is secreted into the duckweed culture medium. 3. The method of claim 1, wherein said nucleotide sequence has at least one at tribute selected from the group consisting of: (a) duckweed-preferred codons in the coding sequence for said .alpha.-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 of 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. 4. The method according to claim 3, wherein said duckweed-preferred codons are Lemna gibba-preferred codons or Lemna minor-preferred codons. 5. The method according to claim 4, 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. 6. The method according to claim 3, wherein said operably linked nucleotide sequence comprising said plant intron is the sequence set forth in SEQ ID NO:1. 7. A method of producing biologically active .alpha.-2b-interferon, comprising the steps of: (a) culturing 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 .alpha.-2b-interferon, and wherein said biologically active .alpha.-2b-interferon is encoded by a nucleotide sequence that has been modified for enhanced expression in duckweed, and (b) collecting said biologically active .alpha.-2b-interferon from said duckweed plant culture or said duckweed nodule culture. 8. The method of claim 7, 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 .alpha.-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. 9. The method according to claim 8, wherein said duckweed-preferred codons are Lemna gibba-preferred codons or Lemna minor-preferred codons. 10. The method according to claim 9, wherein the coding sequence comprises betwee 70% and 100% Lemna gibba-preferred codons or Lemna minor-preferred codons. 11. The method according to claim 8, wherein said operably linked nucleotide sequence comprising said plant intron is the sequence set forth in SEQ ID NO:1. 12. The method according to claim 1, wherein said .alpha.-2b-interferon is a human .alpha.-2b-interferon. 13. The method according to claim 12, wherein said human .alpha.-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. 14. The method according to claim 1, wherein the biologically active .alpha.-2b-interferon variant 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. 15. The method according to claim 1, wherein said signal peptide is selected from the group consisting of: (a) the human .alpha.-2b-interferon signal peptide, (b) the Arabidopsis thaliana chitinase signal peptide; (c) the rice .alpha.-amylase signal peptide; (d) the modified rice .alpha.-amylase peptide; (e) a duckweed signal peptide; and (f) a signal peptide native to the biologically active recombinant polypeptide. 16. The method according to claim 15, wherein the signal peptide is the rice .alpha.-amylase signal peptide having the sequence set forth in SEQ ID NO:3. 17. The stably transformed duckweed plant culture or duckweed nodule culture according claim 1.

Details for Patent 6,815,184

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2020-07-31
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2020-07-31
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2020-07-31