Expression of polypeptides in E. coli under control of the E. coli MDH-gene promoter

An expression vector for expressing in E. coli a polypeptide other than E. coli malate dehydrogenase coded for by a DNA coding sequence, said vector comprising a DNA sequence coding for said polypeptide and including an initiation codon wherein said DNA sequence is operatively linked to an upstream sequence located upstream of the initiation codon and which is capable of controlling expression of said polypeptide, wherein said upstream sequence is a variant of the 285 base pair sequence

• GCA TGC AAA TTC TGC TTA AAA GTA AAT TAA TTG TTA TCA AAT TGA TGT TGT TTT GGC TGA ACG GTA GGG TAT ATT GTC ACC ACC TGT TGG AAT GTT GCG CTA ATG CAT AAG CGA CTG TTA ATT ACG TAA GTT AGG TTC CTG ATT ACG GCA ATT AAA TGC ATA AAC GCT AAA CTT GCG TGA CTA CAC ATT CTT GAG ATG TGG TCA TTG TAA ACG GCA ATT TTG TGG ATT AAG GTC GCG GCA GCG GAG CAA CAT ATC TTA GTT TAT CAA TAT AAT AAG GAG TTT CAT (SEQ ID NO:3),
• wherein said variant has 95% homology with said 285 base pair sequence, and wherein if the initiation codon of the DNA sequence is not ATG it is changed to ATG.

An expression vector for expressing in E. coli a polypeptide other than E. coli malate dehydrogenase coded for by a DNA coding sequence, said vector comprising a DNA sequence coding for said polypeptide and including an initiation codon wherein said DNA sequence is operatively linked to an upstream sequence located upstream of the initiation codon and which is capable of controlling expression of said polypeptide, wherein said upstream sequence is a variant of the 285 base pair sequence

• GCA TGC AAA TTC TGC TTA AAA GTA AAT TAA TTG TTA TCA AAT TGA TGT TGT TTT GGC TGA ACG GTA GGG TAT ATT GTC ACC ACC TGT TGG AAT GTT GCG CTA ATG CAT AAG CGA CTG TTA ATT ACG TAA GTT AGG TTC CTG ATT ACG GCA ATT AAA TGC ATA AAC GCT AAA CTT GCG TGA CTA CAC ATT CTT GAG ATG TGG TCA TTG TAA ACG GCA ATT TTG TGG ATT AAG GTC GCG GCA GCG GAG CAA CAT ATC TTA GTT TAT CAA TAT AAT AAG GAG TTT CAT (SEQ ID NO:3),
• wherein said variant sequence differs from said 285 base pair sequence by between 1 and 10 deletions, insertions and/or substitutions, and wherein if the initiation codon of the DNA sequence is not ATG it is changed to ATG.

An expression vector according to claim, 7 wherein said variant sequence differs from said 285 base pair sequence by not more than 5 deletions, insertions and/or substitutions.

An expression vector according to claim, 10 wherein the promoter (i) has the following base sequence:

• TTGTAAACGGCAATTTTGTGGATTAAGGT (SEQ ID NO:5).

An expression vector for expressing in E. coli a polypeptide other than E. coli malate dehydrogenase coded for by a DNA coding sequence, said vector comprising (1) an E. coli mdh gene promoter having the sequence,

• TTGTAAACGGCAATTTTGTGGATTAAGGT (SEQ ID NO:5)
• and (2) a restriction enzyme site arranged in relation to said promoter so as to allow introduction of the DNA coding sequence into the vector whereby said coding sequence is operatively linked to the promoter, and wherein if the initiation codon of the DNA sequence is not ATG it is changed to ATG.

A recombinant DNA molecule comprising a DNA coding sequence and including an initiation codon operatively linked to an upstream sequence located upstream of the initiation codon, characterised in that the DNA coding sequence codes for a polypeptide other than E. coli malate dehydrogenase and said upstream sequence consists of the 285 base pair sequence

• GCA TGC AAA TTC TGC TTA AAA GTA AAT TAA TTG TTA TCA AAT TGA TGT TGT TTT GGC TGA ACG GTA GGG TAT ATT GTC ACC ACC TGT TGG AAT GTT GCG CTA ATG CAT AAG CGA CTG TTA ATT ACG TAA GTT AGG TTC CTG ATT ACG GCA ATT AAA TGC ATA AAC GCT AAA CTT GCG TGA CTA CAC ATT CTT GAG ATG TGG TCA TTG TAA ACG GCA ATT TTG TGG ATT AAG GTC GCG GCA GCG GAG CAA CAT ATC TTA GTT TAT CAA TAT AAT AAG GAG TTT CAT (SEQ ID NO:3), and wherein if the initiation codon of the DNA sequence is not ATG it is changed to ATG.

A process for producing a polypeptide by expressing a DNA coding sequence, which process comprises culturing a transformed microorganism of the species E. coli wherein (i) said transformed microorganism includes a plasmid comprising said DNA sequence, (ii) the initiation codon of said DNA sequence is changed to ATG, (iii) said DNA sequence is operatively linked to a promoter which is an E. coli mdh promoter and (iv) said polypeptide is expressed such that it forms between 15 and 60% of the soluble cell protein.

A process for producing protein L by expressing DNA sequence coding for protein L which process comprises culturing a transformed microorganism of the species E. coli, wherein (i) said transformed microorganism includes a plasmid comprising said DNA sequence, (ii) if the initiation codon of said DNA sequence is not ATG then it is changed to ATG, (iii) said DNA sequence is operatively linked to a promoter which is an E. coli mdh promoter and (iv) said protein L is expressed such that it forms about 15% of the soluble cell protein.

A process for producing protein G by expressing a DNA sequence coding for protein G which process comprises culturing a transformed microorganism of the species E. coli wherein (i) said transformed microorganism includes a plasmid comprising said DNA sequence, (ii) if the initiation codon of said DNA sequence is not ATG then it is changed to ATG, (iii) said DNA sequence is operatively linked to a promoter which is an E. coli mdh promoter and (iv) said protein G is expressed such that it forms about 25% of the soluble cell protein.