Compositions and methods for protein isolation

Abstract

The invention provides for polynucleotides and vectors comprising at least two tag sequences. The invention also provides for polynucleotides and vectors comprising a streptavidin binding peptide sequence and a calmodulin binding peptide sequence. The invention also provides for polynucleotides and vectors wherein a gene of interest is fused in frame to at least two tag sequences, for example, a streptavidin binding peptide sequence and a calmodulin binding peptide sequence. The invention also provides for the chimeric proteins encoded by these polynucleotides. The invention also provides for methods of using the polynucleotides of the invention for detecting and/isolating protein complexes or identifying a binding partner for a protein of interest.

1 - 16 . (canceled) 
     
     
         17 . A method of detecting or isolating one or more binding partners for a protein encoded by a gene of interest, comprising the steps: cloning a gene sequence of interest into a vector, wherein said gene sequence of interest is fused in frame with at least two different tag sequences, and wherein one of said two tag sequences encodes streptavidin binding peptide having the amino acid sequence presented in  FIG. 1 , introducing said vector into a cell that comprises at least one candidate binding partner for said protein product of said gene of interest; allowing said protein product of said gene sequence of interest and said candidate binding partner to form a complex in the cell; isolating said complex by a) lysing the cells; and b) performing at least one round of affinity purification, and detecting said protein complex. 
     
     
         18 . A method of detecting or isolating one or more binding partners for a protein encoded by a gene sequence of interest, comprising the steps: cloning a gene sequence of interest into a vector, wherein said gene sequence of interest is fused in frame with at least two different tag sequences, and wherein one of said two tag sequences encodes streptavidin binding peptide, and wherein one of said two tag sequences encodes calmodulin binding peptide; introducing said vector into a cell that comprises at least one candidate binding partner for said protein product of said gene sequence of interest; allowing said protein product of said gene of interest and said candidate binding partner to form a complex in the cell; isolating said complex by a) lysing the cells; and b) performing at least one round of affinity purification, and detecting said protein complex. 
     
     
         19 . The method of  claim 17  wherein said cell comprises a vector that expresses at least one candidate binding partner for said protein product of interest. 
     
     
         20 . The method of  claim 17  wherein said candidate binding partner for said protein product of interest comprises a tag. 
     
     
         21 - 27 . (canceled) 
     
     
         28 . The method of  claim 18  wherein said cell comprises a vector that expresses at least one candidate binding partner for said protein product of interest. 
     
     
         29 . The method of  claim 18  wherein said candidate binding partner for said protein product of interest comprises a tag.