Method for construction and use of
View Patent ↗Methods and compositions are provided relating to production of recombinant protein in yeast. A modified P LAC4 is described where one or more mutations may be introduced into the Pribnow box-like sequences in the promoter. The modified promoter when placed upstream of a target gene in a vector causes a significant reduction of target gene expression in transformed bacteria but produces efficient expression of the target gene in yeast.
1. A method of producing a recombinant protein in yeast cells, comprising:
(a) obtaining a vector into which a gene encoding the target protein has been inserted, the vector further comprising: a modified P LAC4 having a mutation in one or more Pribnow box-like sequences wherein the modification results in a significant reduction in gene expression when the vector is cloned in bacteria;
(b) transforming yeast cells with the vector; and
(c) producing an effective amount of the recombinant protein in the yeast cells.
2. A method according to claim 1 , wherein the one or more Pribnow box-like sequences are PBI, PBII and PBIII.
3. A method according to claim 1 , wherein the mutation is in two or more Pribnow box-like sequences.
4. A method according to claim 1 , wherein the modified P LAC4 has one or more mutations in a first region of the promoter corresponding to nucleotides −198 to −212.
5. A method according to claim 1 , wherein the modified P LAC4 has one or more mutations in a second region of the promoter corresponding to nucleotides −133 to −146.
6. A method according to claim 1 , wherein nucleotides −1 to −283 in the modified P LAC4 are substituted by nucleotides −1 to −283 of a phosphoglycerate kinase promoter from S. cerevisiae.
7. A method according to claim 1 , wherein the vector in the transformed yeast cells is an episomal plasmid.
8. A method according to claim 1 , wherein the vector in the transformed yeast cells is an integrative plasmid.
9. A vector, comprising: a modified P LAC4 promoter having a mutation in one or more Pribnow box-like sequences operatively linked to a gene encoding a target protein.
10. A vector according to claim 9 , wherein the one or more Pribnow box-like sequences are PBI, PBII and PBIII.
11. A vector according to claim 9 , wherein the mutation is in two or more Pribnow box-like sequences.
12. A vector according to claim 9 , wherein the modified P LAC4 has one or more mutations in a first region of the promoter corresponding to nucleotides −198 to −212.
13. A vector according to claim 9 , wherein the modified P LAC4 has one or more mutations in a second region of the promoter corresponding to nucleotides −133 to −146.
14. A vector according to claim 9 , wherein nucleotides −1 to −283 in the modified P LAC4 are substituted by nucleotides −1 to −283 of a phosphoglycerate kinase promoter from S. cerevisiae.
15. A host yeast cell comprising a vector of claim 9 .
16. A kit, comprising: a vector according to claim 9 ; and optionally, competent yeast cells and instructions for use.