Claims for Patent: 8,980,580

Summary for Patent: 8,980,580

Title:	Heterologous expression of fungal polyketide synthetic gene in yeast and a method of preparing a compound produced by a protean encoded by the polyketide synthetic gene by the heterologous expression
Abstract:	The present invention relates to a method of removing an intron contained in a gene from a eukaryotic gene, and linking only the exon sequences to prepare an expression vector comprising the linked sequences. Specifically, the invention relates to a method of preparing an expression vector containing linked exon sequences comprising amplifying exon sequences by PCR as one or more fragments from a giant fungal gene containing an intron, and linking the fragments together with a restriction enzyme-treated vector using the gap repair cloning method; a method of preparing an expression vector containing a full-length cDNA sequence by synthesizing and linking cDNA fragments from a fungal giant gene; a transformant having introduced therein an expression vector prepared by the method; a protein produced by the transformant; and a method of preparing a compound produced by the protein using the expression vector.
Inventor(s):	Watanabe; Kenji (Shizuoka, JP), Moriya; Hisao (Okayama, JP)
Assignee:	Shizuoka Prefecture Public University Corporation (Shizukoa, JP) National University Corporation Okayama University (Okayama, JP)
Application Number:	13/816,826
Patent Claims:	1. A method of preparing a compound produced by a protein encoded by the gene or genome sequence of the presumed gene containing an intron by using an expression vector prepared by a method of preparing an expression vector by linking exon sequences of a eukaryotic gene containing an intron or from the genome sequence of a presumed eukaryotic gene containing an intron to form the expression vector containing the linked sequences, said method comprising the steps of: (a) amplifying exon sequences from a genome extracted from a eukaryote by PCR to prepare multiple fragments, wherein the forward primer used in the PCR has, in order from the 5' end to the 3' end, a sequence complementary to the sequence of the 3' terminal part of the sense strand of a fragment to which the amplified fragment is to be linked, or a sequence complementary to the sequence of the 3' terminal part of the sense strand of a restriction enzyme-treated terminal part of the vector, and a sequence complementary to the sequence of the 5' terminal part of the sense strand of the fragment to be amplified, and wherein the reverse primer has, in order from the 5' end to the 3' end, a sequence complementary to the sequence of the 3' terminal part of the antisense strand of a fragment to which the amplified fragment is to be linked, or a sequence complementary to the sequence of the 3' terminal part of the antisense strand of a restriction enzyme-treated terminal part of the vector, and a sequence complementary to the sequence of the 5' terminal part of the antisense strand of the fragment to be amplified, whereby a sequence homologous to a terminal part of a fragment to be linked to the fragment to be amplified or a sequence homologous to a restriction enzyme-treated terminal part of the vector are added to the end of the fragment to be amplified; and (b) simultaneously transforming a budding yeast or fission yeast with the fragments obtained in the step (a) and a restriction enzyme-treated vector to obtain the expression vector containing fragments linked to the fragments and fragments linked to the vector that are joined via homologous recombination, wherein the gene or genome sequence of the presumed gene encodes a polyketide synthase gene or nonribosomal peptide synthetase gene, and wherein the linked sequence is a polynucleotide comprising the nucleotide sequence of SEQ ID NOs:15 to 21, 29 or 47. 2. The method according to claim 1, comprising culturing a transformant having an introduced expression vector, and collecting the compound from the culture medium or the transformant. 3. A method of preparing a compound produced by a protein encoded by the gene or genome sequence of the presumed gene containing an intron by using an expression vector prepared by a method of preparing an expression vector comprising a full-length cDNA sequence from a eukaryotic gene containing an intron or of the genome sequence of a presumed eukaryotic gene containing an intron, said method comprising the steps of: (a) synthesizing cDNA fragments from mRNA extracted from a eukaryote and amplifying the cDNA fragments by PCR, wherein the forward primer used in the PCR has, in order from the 5' end to the 3' end, a sequence complementary to the sequence of the 3' terminal part of the sense strand of a fragment to which the amplified fragment is to be linked, or a sequence complementary to the sequence of the 3' terminal part of the sense strand of a restriction enzyme-treated terminal part of the vector, and a sequence complementary to the sequence of the 5' terminal part of the sense strand of the fragment to be amplified, and wherein the reverse primer has, in order from the 5' end to the 3' end, a sequence complementary to the sequence of the 3' terminal part of the antisense strand of a fragment to which the amplified fragment is to be linked, or a sequence complementary to the sequence of the 3' terminal part of the antisense strand of the restriction enzyme-treated terminal part of the vector, and a sequence complementary to the sequence of the 5' terminal part of the antisense strand of the fragment to be amplified, whereby a sequence homologous to a terminal part of a fragment to be linked to the fragment to be amplified or a sequence homologous to a restriction enzyme-treated terminal part of the vector are added to the end of the fragment to be amplified; and (b) simultaneously transforming a budding yeast or fission yeast with the cDNA fragments obtained in the step (a) and a restriction enzyme-treated vector to obtain the expression vector containing fragments linked to the fragments and fragments linked to the vector that are joined via homologous recombination, wherein the gene or genome sequence of the presumed gene encodes a polyketide synthase gene or nonribosomal peptide synthetase gene, and wherein the linked sequence is a polynucleotide comprising the nucleotide sequence of SEQ ID NOs:15 to 21, 29 or 47. 4. The method according to claim 3, comprising culturing a transformant having an introduced expression vector, and collecting the compound from the culture medium or the transformant.

Details for Patent 8,980,580

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		2030-08-13
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2030-08-13
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2030-08-13