Prokaryotic collage-like proteins and uses thereof

Abstract

The present invention provides recombinant triple helical proteins or collagen-like proteins comprising a prokaryotic protein or one or more domains of a prokaryotic protein comprising a collagen-like peptide sequence of repeated Gly-Xaa-Yaa triplets and, optionally, one or more domains from a mammalian collagen. Also provided are expression vectors and host cells containing the expression vectors to produce these recombinant proteins and methods of producing the same. Additionally, antibodies are provided that are directed against a recombinant collagen-like protein that, preferably, binds an integrin. Furthermore, a method of screening for potential therapeutic compounds that inhibit the integrin-binding or integrin-interacting activities of recombinant collagen-like proteins.

1 . An expression vector, comprising:
 a DNA sequence encoding a recombinant collagen-like protein comprising one or more domains having one or more of the amino acid sequences of SEQ ID NOS: 1-42, wherein the protein thereby forms a triple helical structure.   
     
     
         2 . The expression vector of  claim 1 , wherein the encoded recombinant collagen-like protein further comprises one or more triple helical domains of a mammalian collagen as a fusion protein. 
     
     
         3 . The expression vector of  claim 2 , wherein the mammalian collagen is human collagen. 
     
     
         4 . A host cell comprising and expressing the expression vector of  claim 1 . 
     
     
         5 . The host cell of  claim 4 , wherein said host cell is  Escherichia coli, Streptococcus  or  Bacillus.    
     
     
         6 . A method of producing a collagen-like protein, comprising:
 introducing into a prokaryotic host cell the expression vector of  claim 1 ;   culturing the prokaryotic host cell under conditions suitable to express the protein;   isolating said expressed protein; and   purifying said collagen-like protein, thereby producing the collagen-like protein.   
     
     
         7 . The method of  claim 6 , wherein the host cell is  Escherichia coli, Streptococcus  or  Bacillus.    
     
     
         8 . A method of producing a gelatin-like material, comprising:
 introducing into a prokaryotic host cell an expression vector of  claim 1 ;   culturing the prokaryotic host cell under conditions suitable to express said protein; and   heating the expressed protein thereby producing said gelatin-like material.   
     
     
         9 . The method of  claim 8 , wherein the host cell is  Escherichia coli, Streptococcus  or  Bacillus.    
     
     
         10 . An antibody directed against a recombinant streptococcal collagen-like (rScl) protein that binds to α 2 β 1  integrin or to an α 2 -I domain thereof. 
     
     
         11 . The antibody of  claim 10 , wherein said rScl protein is p176. 
     
     
         12 . A method for screening for therapeutic compounds that inhibit a Streptococcal collagen-like protein (Scl) binding to or interacting with an integrin, comprising:
 measuring Scl binding to or interaction with an α 2 β 1  integrin or an α 2 -I domain therein in the presence of a test compound;   measuring Scl binding to or interaction with the integrin in the absence of the test compound in a control sample;   comparing binding of or interaction with the integrin to the Scl in the presence of the test compound with binding or interaction in the control sample; and   correlating a reduction in binding of or interaction with the integrin to the Scl in the presence of the test compound compared to control with inhibition of integrin binding to or interaction with the Scl by said test compound, thereby screening for the therapeutic compound.   
     
     
         13 . The method of  claim 12 , wherein the Scl is P176.