US 5,635,371 AGranted

Chemical method for selective modification of the N- and/or C-terminal amino acid .alpha.-carbon reactive group of a recombinant polypeptide or a portion thereof

AssigneeBioNebraska, Inc.

InventorsJay Stout, Fred W. Wagner, Thomas R. Coolidge, Bart Holmquist

Life of this patent

Estimating the term…

Loading patent…

Abstract

The invention provides for a chemical method for preparing a recombinant single copy polypeptide or a portion thereof with a modified terminal amino acid .alpha.-carbon reactive group selected from the group consisting of N-terminal .alpha.-amine, C-terminal .alpha.-carboxyl, and a combination thereof. The steps of the method involve forming the recombinant single copy polypeptide or a portion thereof so that the single copy polypeptide is protected with one or more biologically added protecting groups at the N-terminal .alpha.-amine, C-terminal .alpha.-carboxyl. The recombinant single copy polypeptide can then be reacted with up to three chemical protecting agents to selectively protect reactive side chain groups and thereby prevent side chain groups from being modified. The recombinant single copy polypeptide can be cleaved with at least one cleavage reagent specific for the biological protecting group to form an unprotected terminal amino acid .alpha.-carbon reactive group. The unprotected terminal amino acid .alpha.-carbon reactive group is modified with at least one chemical modifying agent. The side chain protected terminally modified single copy polypeptide is then deprotected at the side chain groups to form a terminally modified recombinant single copy polypeptide. The number and sequence of steps in the method can be varied to achieve selective modification at the N- and/or C-terminal amino acid of a recombinantly produced polypeptide.

Claims17

Dependent claims

A method for selectively modifying a recombinantly produced polypeptide only at a terminal .alpha.-carbon reactive group selected from the group consisting of N-terminal .alpha.-amine, C-terminal .alpha.-carboxyl, and a combination thereof comprising:

modifying the recombinantly produced polypeptide with a biologically added protecting group on one or both terminal .alpha.-carbon reactive groups by modifying a DNA sequence encoding the recombinant polypeptide by adding a DNA sequence encoding the biologically added protecting group at the 5' and/or 3' end of the DNA sequence coding for the recombinant polypeptide, wherein the biologically added protecting group is a group that has at least one cleavage sequence that provides for cleavage and removal of the biologically added protecting group and generation of an unprotected terminal reactive .alpha.-carbon group on the recombinantly produced polypeptide upon removal, and wherein the recombinantly produced polypeptide has reactive side chain groups selected from the group consisting of .epsilon.-amine, hydroxyl, .beta.-carboxyl, .gamma.-carboxyl, thiol, and a combination thereof;
conducting the following reacting and removing steps in any order to produce a side chain protected recombinant polypeptide having at least one unprotected terminal amino acid .alpha.-carbon reactive group:
(i) reacting the recombinantly produced polypeptide with up to three chemical protecting agents to selectively protect a reactive side chain group selected from the group consisting of .epsilon.-amine, hydroxyl, .beta.-carboxyl, .gamma.-carboxyl, thiol, and a combination thereof;
(ii) removing the biologically added protecting group with at least one cleavage reagent specific for the biologically added protecting group to form an unprotected terminal amino acid .alpha.-carbon reactive group;
modifying the unprotected terminal amino acid .alpha.-carbon reactive group with at least one chemical modifying agent to form a terminally modified side chain protected recombinant polypeptide;
and
deprotecting the terminally modified side chain protected recombinant polypeptide to form the terminally modified recombinant polypeptide.

4 more elements

A method for selectively modifying a recombinantly produced polypeptide only in a terminal .alpha.-carbon reactive group selected from the group consisting of N-terminal .alpha.-amine, C-terminal .alpha.-carboxyl, and a combination thereof from a recombinantly produced multicopy polypeptide, comprising:

modifying the recombinantly produced multicopy polypeptide with a biologically added protecting group on a terminal .alpha.-carbon reactive group on each copy of the recombinant polypeptide by modifying a DNA sequence encoding the recombinant multicopy polypeptide by adding a DNA sequence encoding the biologically added protecting group at the 5' or 3' end of the DNA sequence encoding each copy of the multicopy polypeptide, wherein the biologically added protecting group has at least one cleavage sequence that provides for cleavage and removal of the biologically added protecting group and generation of terminal .alpha.-carbon reactive group upon removal, and wherein the recombinant polypeptide includes a .epsilon.-amine, hydroxyl, .beta.-carboxyl, .gamma.-carboxyl or thiol reactive side chain group;
conducting the following reacting and removing steps in any order to produce a side chain protected recombinant polypeptide having at least one unprotected terminal amino acid .alpha.-carbon reactive group:
reacting the reactive side chain group with a chemical protecting agent to selectively protect the reactive side chain group;
removing the biologically added protecting group with at least one cleavage reagent specific for the biologically added protecting group to form an unprotected terminal amino acid .alpha.-carbon reactive group on each copy of the recombinant polypeptide;
modifying the unprotected N-terminal .alpha.-amine or C-terminal .alpha.-carboxyl group with a chemical modifying agent to form a modified N-terminal .alpha.-amine or C-terminal .alpha.-carboxyl side chain protected recombinant polypeptide;
and
deprotecting the N-terminal .alpha.-amine modified or C-terminal .alpha.-carboxyl side chain protected recombinant polypeptide to form a terminally modified recombinant polypeptide.

4 more elements

Dependencies are inferred from the claim text.

Facts

Application: 08/294,434Filed 23 Aug 1994 · continuation
Priority: 13 Jul 1992
Granted: 3 Jun 1997
Term ends: Not estimatedThe source records don’t support an estimate.

Classification18 codes

C07K 14/60
Hormones: growth hormone-releasing factor [GH-RF], i.e. somatoliberin
C07K 1/003
General methods for the preparation of peptides , i.e. processes for the organic chemical preparation of peptides or proteins of any length: by…
C07K 1/006
General methods for the preparation of peptides , i.e. processes for the organic chemical preparation of peptides or proteins of any length: of…
C07K 1/1072
By chemical modification of precursor peptides: by covalent attachment of residues or functional groups