Expression vector for improved production of polypeptides in yeast

Abstract

A new expression vector for the production of a polypeptide in yeast. The vector includes a sequence coding for the polypeptide and other sequences allowing expression of the polypeptide only in yeast. The other sequences lack any non-yeast sequences. Other embodiments include a yeast strain comprising such a vector, a method for the production of the vector, a method for the production of the yeast strain by transformation of a yeast strain with the new vector, and a method for the production of a polypeptide in the transformed yeast strain by fermentation thereof followed by isolation of the polypeptide.

What is claimed is:  
     
         1 . An isolated expression vector construct for the production of a polypeptide in yeast, the expression vector comprising (1) a DNA sequence encoding for the polypeptide and (2) DNA sequences allowing for the expression of the polypeptide in yeast, wherein the expression vector, except for any DNA sequences encoding for non-yeast polypeptides, lacks any non-yeast DNA sequences, the expression vector being obtainable by a process comprising: 
 (A) isolating genomic DNA from a yeast;    (B) isolating a chromosomal fragment carrying a Cu/Zn SOD gene with upstream and downstream regions;    (C) subcloning said chromosomal fragment into a plasmid pCRII;    (D) excising and purifying a fragment carrying the Cu/Zn SOD gene using restriction enzymes BamHI and SalI;    (E) subcloning the purified fragment into the BamHI-SalI sites of vector pEMBLyex4 to produce the vector pEMBL-SOD 374 with the Cu/Zn SOD gene and an upstream sequence;    (F) deleting the polylinker multiple cloning site from a sample of vector pEMBL-SOD 374 by double digestion of the polylinker with the enzymes SstI and HindIII;    (G) isolating a HindIII-SacI fragment with the yeast and bacterial sequences of the vector pEMBL without the polylinker;    (H) purifying and concentrating the HindIII-SacI fragment;    (I) double digestion of a sample clone pEMBL-SOD 374 carrying the Cu/Zn SOD gene with enzymes EcoRI and SalI to isolate a DNA fragment carrying the entire open reading frame of the Cu/Zn SOD gene together with upstream and downstream sequences;    (J) producing blunt ends on the EcoRI-SalI fragment;    (K) subcloning the EcoRI-SalI fragment into the HindIII-SacI fragment;    (L) isolating clones from the ligation by random screening that have the vector pEMBL-SOD without the multiple cloning site sequences;    (M) digesting pEMBL-SOD without multiple cloning site sequences with StuI and NruI;    (N) isolating the remaining the fragment carrying only yeast sequences; and    (O) ligating the ends of the fragment to produce vector pEMBL-SOD.    
     
     
         2 . The expression vector of  claim 1 , wherein the yeast is  Saccharomcyces cerevisiae.    
     
     
         3 . A method for production of an expression vector, the method comprising: 
 isolating a first chromosomal fragment from a yeast, wherein the fragment comprises a Cu/Zn SOD gene with upstream and downstream regions;    subcloning the first fragment into a plasmid pCRII;    excising a second fragment carrying the Cu/Zn SOD gene from the plasmid pCRII carrying the first fragment;    subcloning the second fragment into the BamI-SalI sites of a vector pEMBLyex4 to obtain a vector pEMBL-SOD 374;    isolating a Hind III-Sac I fragment from the vector pEMBL-SOD 374, wherein the Hind III-Sac I fragment comprises yeast and bacterial sequences of the vector pEMBLyex4 but not does not include the polylinker multiple cloning site;    isolating an EcoRI-SalI fragment from a sample of clone pEMBL-SOD 374, wherein the EcoRI-SalI fragment comprises the entire open reading frame of the Cu/Zn SOD gene and at least a portion of the upstream and downstream sequences;    subcloning the EcoRI-Sal I fragment into the Hind III-Sac I fragment to obtain a third fragment;    isolating clones of the third fragment, wherein the isolated clones lack the multiple cloning site sequences;    digesting an isolated clone to delete all non-yeast bacterial sequences to obtain a fourth fragment comprising the marker Leu-2d and the Cu/Zn SOD gene; and    rejoining ends of the fourth fragment to produce a vector comprising the Cu/Zn SOD gene, the Leu-2d marker, the origin of replication of the two-micron vector, and the entire yeast expression hybrid promoter cassette UAS GAL/CYC, wherein the vector lacks bacterial sequences and the multiple cloning site.    
     
     
         4 . The method of  claim 3 , wherein the yeast is  Saccharomcyces cerevisiae.    
     
     
         5 . The method of  claim 4 , further comprising transforming a yeast strain with the expression vector that lacks, except for any DNA sequences coding for non-yeast polypeptides, any non-yeast DNA sequences.  
     
     
         6 . The method of  claim 5 , wherein the yeast strain is  Saccharomcyces cerevisiae.    
     
     
         7 . The method of  claim 6 , wherein the strain is GRF18.  
     
     
         8 . The method of  claim 3 , wherein the vector lacks, except for any DNA sequences coding for non-yeast polypeptides, any non-yeast DNA sequences.