Stabilized proteins

Abstract

The invention described herein comprises methods for stabilizing polypeptides and polypeptide complexes, and the polypeptides and polypeptide complexes stabilized using the methods. To achieve stabilization, a cross-link reaction is controlled such that polypeptides and polypeptide complexes maintain their original functionality. In one embodiment, the invention provides a method for the identification of amino acid residues which, when cross-linked, are least disruptive to the structure and function of the polypeptide or polypeptide complex. In another embodiment, the invention provides a method for mutagenesis of identified residues to further control the cross-link reaction. Polypeptides and polypeptide complexes so stabilized can be utilized under a wide variety of physiological and non-physiological conditions. Further, the cross-link methodology disclosed herein may preclude the need for addition of exogenous structures to engineered proteins and complexes, such as peptide linkers that could be immunogenic and/or significantly decrease efficacy. In another embodiment, the invention provides a method for statistical analysis of databases of structural and/or sequence information available for polypeptides and polypeptide complexes to be stabilized. The statistical analysis identifies suitable residue pairs which are least likely to be disruptive of structure and function when cross-linked. Further, in a polypeptide chain or chains to be cross-linked, potentially undesirable reactive side-chains may be masked and protected, or altered using site-directed mutagenesis, e.g., to introduce a maximally conservative point mutation that will not support the cross-link reaction. The cross-link reaction conditions may also be adjusted to prevent undesired cross-links or other undesired side-effects. At residues identified as desirable positions for cross-linking, reactive side-chains may be introduced by site-directed mutagenesis, and the cross-link reaction is carried out using the conditions identified above.

1 . A method for making a stabilized protein or fragment thereof comprising: (a) selecting one or more residue pairs in a polypeptide chain or chains for cross-linking using one or more statistical criteria; and (b) cross-linking the residue pairs.  
     
     
         2 . The method of  claim 1 , wherein the stabilized protein or fragment is selected from the group consisting of a hormone, a receptor, a growth factor, an enzyme and an antibody.  
     
     
         3 . The method of  claim 2 , wherein the enzyme is a lipase or the antibody fragment is an Fv fragment.  
     
     
         4 . The method of  claim 1 , wherein the one or more statistical criteria used for selection of residue pairs in step (a) are selected from the group consisting of statistical filter one through statistical filter six.  
     
     
         5 . The method of  claim 1 , wherein tyrosine residues are cross-linked.  
     
     
         6 . The method of  claim 6 , wherein cross-linking is catalyzed by a catalyst selected from the group consisting of polyhistidine, Gly-Gly-His and metalloporphyrin.  
     
     
         7 . The method of  claim 6 , wherein the cross-linked tyrosine residues are introduced into the stabilized protein complex prior to cross-linking by recombinant nucleic acid methods.  
     
     
         8 . A method for identifying a residue pair in a polypeptide chain or chains that, following substitution with tyrosine and cross-linking, is least likely to be disruptive of overall protein structure, comprising applying one or more statistical criteria selected from the group consisting of statistical filter one through statistical filter six.  
     
     
         9 . A protein cross-linked by the method of  claim 1 .  
     
     
         10 - 20 . (canceled)