Process for producing protein A-like protein with use of
View Patent ↗The present invention relates to an efficient and economical process for producing a protein A-like protein. Hosts such as Escherichia coli and Bacillus subtilis have been used in the production of a protein A-like protein using a genetic recombination technique and however, their low productivity has been a big cause of high cost. Thus, it has been desired strongly to immediately establish a technique enabling the inexpensive, large-scale production of a protein A-like protein using recombinant DNA techniques other than Escherichia coli and Bacillus subtilis. The present invention provides a process for producing a protein A-like protein in large amounts, for example, a process comprising allowing a recombinant Brevibacillus genus bacterium to express and secrete the protein in large amounts into a culture solution and separating and collecting the accumulated protein A-like protein from the culture solution.
1. An isolated DNA sequence comprising:
(a) a DNA sequence encoding a Staphylococcus aureus protein A or a variant protein thereof having at least 90% amino acid identity to SEQ ID NO: 2, wherein said variant protein has immunoglobulin-binding activity; and
(b) a promoter which is operatively linked to said DNA sequence encoding a Staphylococcus aureus protein A or a variant protein thereof, wherein said promoter has a promoter activity when said isolated DNA sequence is transformed into a Brevibacillus genus bacterium wherein the promoter is a promoter of a cell wall protein of a Brevibacillus genus bacterium, and wherein the isolated DNA sequence further comprises downstream of the promoter, a Shine-Dalgarno sequence which is capable of functioning in a Brevibacillus genus bacterium and a secretion signal peptide-encoding DNA sequence which is capable of functioning in a Brevibacillus genus bacterium.
2. An expression vector comprising a DNA sequence according to claim 1 .
3. A Brevibacillus genus bacterium transformant comprising an expression vector according to claim 2 .
4. The transformant according to claim 3 , wherein the Brevibacillus genus bacterium is selected from the group consisting of a Brevibacillus brevis 47 strain (JCM6285), Brevibacillus brevis 47K strain (FERM BP-2308), Brevibacillus brevis 47-5Q strain (JCM8970), Brevibacillus choshinensis HPD31 strain (FERM BP-1087), and Brevibacillus choshinensis HPD31-OK strain (FERM BP-4573), and mutants derived from these strains.
5. A process for producing a Staphylococcus aureus protein A or a variant protein thereof having at least 90% amino acid identity to SEQ ID NO: 2, wherein said variant protein has immunoglobulin-binding activity, comprising culturing a transformant according to claim 3 and collecting said Staphylococcus aureus protein A or said polypeptide produced and secreted by the transformant.
6. A process for producing an immunoglobulin-adsorbing medium, comprising producing a Staphylococcus aureus protein A or a variant protein thereof having at least 90% amino acid identity to SEQ ID NO: 2, wherein said variant protein has immunoglobulin-binding activity, by a producing process according to claim 5 and immobilizing said Staphylococcus aureus protein A or said polypeptide onto an appropriate base matrix.
7. A process for producing a Staphylococcus aureus protein A or a variant protein thereof having at least 90% amino acid identity to SEQ ID NO: 2, wherein said variant protein has immunoglobulin-binding activity, comprising culturing a transformant according to claim 4 and collecting said Staphylococcus aureus protein A or said polypeptide produced and secreted by the transformant.
8. A process for producing an immunoglobulin-adsorbing medium, comprising producing a Staphylococcus aureus protein A or a protein A-like protein that has at least 90% amino acid residue identity to SEQ ID NO: 2 and has immunoglobulin-binding activity, by a producing process according to claim 7 and immobilizing said Staphylococcus aureus protein A or said polypeptide onto an appropriate base matrix.