Chimeric protein containing an intramolecular chaperone-like sequence
AssigneeTonghua GanTech Biotechnology Ltd.
InventorZhong-Ru Gan
Life of this patent
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Abstract
The present invention relates to a chimeric protein containing an intramolecular chaperone (IMC) like sequence linked to a target protein, preferably an insulin precursor. The present invention also relates to a process for obtaining a correctly folded insulin-precursor-containing chimeric protein, comprising, inter alia, contacting an incorrectly folded chimeric protein containing an IMC like sequence linked to an insulin precursor with at least one chaotropic auxiliary agent. The present invention further relates to an assay for screening an amino acid sequence for the ability to improve folding of an insulin precursor using a chimeric protein containing an IMC like sequence linked to an insulin precursor.
Claims77
A chimeric protein comprising, from N-terminus to C-terminus:
- a) a first peptidyl fragment consisting of an amino acid sequence that has at least 40% identity to a domain containing at least first 20 N-terminal amino acids of human growth hormone (hGH) protein, in which the percentage identity is determined over an amino acid sequence of identical size to the domain of hGH;
- b) an Arg residue, or a Lys residue, or a second peptidyl fragment consisting of at least 2 amino acids in which peptidyl fragment the most C-terminal amino acid residue is an Arg or a Lys residue;
- and
- c) a third peptidyl fragment consisting of an amino acid sequence containing more than two cysteine (Cys) residues which peptidyl fragment is not a portion of hGH protein.
A chimeric protein consisting of the amino acid sequence of SEQ ID NO:
- 6.
A chimeric protein consisting of the amino acid sequence of SEQ ID NO:
- 7.
A process for obtaining a correctly folded first insulin-precursor-containing chimeric protein, comprising contacting an incorrectly folded second insulin-precursor-containing chimeric protein, which said second insulin-precursor-containing chimeric protein consists of an intramolecular chaperone (IMC) like peptidyl fragment separated from the insulin precursor by one or more cleavable amino acid residues, with at least one chaotropic auxiliary agent in an aqueous medium;
- wherein said IMC like peptidyl fragment:
- a) contains from about 20 to about 200 amino acid residues;
- b) is not the insulin precursor or a portion thereof;
- and
- c) improves the insulin precursor folding such that the yield of the correctly folded first insulin-precursor-containing chimeric protein when the incorrectly folded second insulin-precursor-containing chimeric protein is contacted with the chaotropic auxiliary agent is higher than the yield of the correctly folded insulin precursor when the incorrectly folded insulin precursor, which does not contain said IMC like peptidyl fragment, is contacted with the same chaotropic auxiliary agent.
Dependencies are inferred from the claim text.