Basic protein purification tags from thermophilic bacteria
The invention is related to a method for purification of recombinant proteins using highly basic proteins from thermophilic bacteria as purification tags for use in a cation-exchange chromatography purification step. The basic proteins may be ribosomal proteins. The recombinant proteins are expressed in eukaryotic or prokaryotic host cells. The purification tag will typically have a pl above about 9 and comprise from about 15 to about 250 amino acid residues.
1. A method for purification of a recombinant target protein expressed in a eukaryotic or prokaryotic host cell comprising:
(a) isolating an expressed tagged protein from the host cell, wherein the expressed tagged protein comprises a target protein, the purification tag of SEQ ID NO: 24, and a linker sequence comprising a cleavage site for in vitro cleavage of the target protein from the purification tag;
(b) subjecting the expressed tagged protein to cation-exchange chromatography purification;
(c) cleaving the target protein from the purification tag; and
(d) isolating the target protein.
2. The method according to claim 1 , wherein the linker has from 1 to about 30 amino acid residues.
3. The method according to claim 1 , wherein the linker has from 1 to about 15 amino acid residues.
4. The method according to claim 1 , wherein the linker comprises amino acid residues selected from the group consisting of Pro, Leu and Ala.
5. The method according to claim 1 , wherein the linker comprises a cleavage site which is selected from the group consisting of an enterokinase cleavage site, a Factor Xa cleavage site, a thrombin cleavage site, a Tobacco etcs virus protease cleavage site and a HRV14 3C protease cleavage site.
6. The method according to claim 1 , wherein the linker has a peptide sequence of SEQ ID NO: 9.
7. The method according to claim 1 , wherein the host cell is selected from bacteria or fungi such as Eschericia sp., Bacillus sp., Saccharomyces sp. and, Aspergillus sp.
8. The method according to claim 1 further comprising a heat precipitation step for precipitating thermolabile host cell contaminants before the cation-exchange column in step (b).
9. The method according to claim 1 , wherein the target protein is human hGH or an analogue thereof.
10. The method according to claim 9 , wherein the target protein is hGH-Leu-Ala or Ser-hGH.
11. The method according to claim 8 , wherein from about 30 to about 200 mM NaCl is added before the heat precipitation step.
12. The method of claim 1 wherein cleaving the target protein from the purification tag is accomplished by a suitable processing enzyme.