Insulin analogues with chlorinated amino acids and nucleic acids encoding the same
An insulin analog comprises a B-chain polypeptide incorporating a chlorinated phenylalanine. The chlorinated phenylalanine may be located at position B24. The chlorinated phenylalanine may be para-monochloro-phenylalanine. The analog may be of a mammalian insulin, such as human insulin. A nucleic acid encodes such an insulin analog. The chlorinated insulin analogs retain significant activity. A method of treating a patient comprises administering a physiologically effective amount of the insulin analog or a physiologically acceptable salt thereof to a patient. Chlorine substitution-based stabilization of insulin may reduce fibrillation and thereby enhance the treatment of diabetes mellitus in regions of the developing world lacking refrigeration.
1. A method of incorporating a chlorinated phenylalanine into
an insulin B-chain polypeptide, the method comprising:
expressing a nucleic acid encoding the insulin B-chain polypeptide,
wherein the chlorinated phenylalanine is encoded by a stop codon,
wherein the nucleic acid is expressed in conjunction with a suppressor tRNA and a corresponding tRNA synthetase which incorporates the chlorinated phenylalanine in response to the stop codon,
wherein the chlorinated phenylalanine is incorporated into the insulin B-chain polypeptide at position B24 relative to wild type insulin, and
wherein the chlorinated phenylalanine is para-monochloro-phenylalanine.
2. The method of claim 1 wherein the stop codon is TAG.
3. The method of claim 2 , wherein the nucleic acid encoding the insulin B-chain polypeptide comprises SEQ ID NO: 11 or SEQ ID NO: 12.
4. The method of claim 2 , wherein the nucleic acid encoding the insulin B-chain polypeptide comprises nucleotides 61-90 of SEQ ID NO: 11 or nucleotides 61-90 of SEQ ID NO: 12.
5. The method of claim 1 , wherein the nucleic acid is a portion of an expression vector.
6. The method of claim 5 , wherein the expression vector is inserted into a host cell.
7. A method of producing an insulin analogue containing a chlorinated phenylalanine, the method comprising expressing a nucleic acid encoding at least amino acid residues 20-30 of the insulin B-chain polypeptide, wherein the chlorinated phenylalanine encoded by a stop codon and wherein the nucleic acid is expressed in conjunction with a suppressor tRNA and a corresponding tRNA synthetase which incorporates the chlorinated phenylalanine in response to the stop codon, and wherein the chlorinated phenylalanine is para-monochloro-phenylalanine located at position B24 relative to wild type insulin.