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US 5,874,239 AGranted
Biotinylation of proteins
AssigneeAffymax Technologies N.V.
InventorPeter J. Schatz
Life of this patent
Estimating the term…
Abstract
Biotinylation peptides can be fused to other peptides or proteins of interest using recombinant DNA techniques to provide efficient methods for biotinylating the resulting fusion proteins in vivo or in vitro.
Claims11
A method for biotinylating a protein, said method comprising:
- (a) constructing a recombinant DNA expression vector that encodes a fusion protein comprising said protein and a biotinylation peptide wherein said biotinylation peptide comprises an amino acid sequence defined by:
- Leu Xaa.sub.1 Xaa.sub.2 Ile Xaa.sub.3 Xaa.sub.4 Xaa.sub.5 Xaa.sub.6 Lys Xaa.sub.7 Xaa.sub.8 Xaa.sub.9 Xaa.sub.10 (SEQ ID NO:1), where Xaa.sub.1 is any amino acid;
- Xaa.sub.2 is any amino acid other than Leu, Val, Ile, Trp, Phe, or Tyr;
- Xaa.sub.3 is Phe or Leu;
- Xaa.sub.4 is Glu or Asp;
- Xaa.sub.5 is Ala, Gly, Ser, or Thr;
- Xaa.sub.6 is Gln or Met;
- Xaa.sub.7 is Ile, Met, or Val;
- Xaa.sub.8 is Glu, Leu, Val, Tyr, or Ile;
- Xaa.sub.9 is Trp, Tyr, Val, Phe, Leu, or Ile;
- and Xaa.sub.10 is any amino acid other than Asp or Glu, and wherein said biotinylation-peptide is capable of being biotinylated by a biotin ligase at said lysine residue adjacent to Xaa.sub.6 ;
- and is 13 to 50 amino acids in length;
- (b) transforming a recombinant host cell with said vector;
- and
- (c) culturing said host cell in the presence of biotin or a biotin analogue and under conditions such that said fusion protein and a biotinylation enzyme are expressed, resulting in biotinylation of said fusion protein.
12 more elements
A method for biotinylating a protein, said method comprising:
- (a) constructing a recombinant DNA expression vector that encodes a fusion protein comprising said protein and a biotinylation peptide wherein said biotinylation peptide comprises an amino acid sequence defined by:
- Leu Xaa.sub.1 Xaa.sub.2 Ile Xaa.sub.3 Xaa.sub.4 Xaa.sub.5 Xaa.sub.6 Lys Xaa.sub.7 Xaa.sub.8 Xaa.sub.9 Xaa.sub.10 (SEQ ID NO:1), where Xaa.sub.1 is any amino acid;
- Xaa.sub.2 is any amino acid other than Leu, Val, Ile, Trp, Phe, or Tyr;
- Xaa.sub.3 is Phe or Leu;
- Xaa.sub.4 is Glu or Asp;
- Xaa.sub.5 is Ala, Gly Ser, or Thr;
- Xaa.sub.6 is Gln or Met;
- Xaa.sub.7 is Ile, Met, or Val;
- Xaa.sub.8 is Glu, Leu, Val, Tyr, or Ile;
- Xaa.sub.9 is Trp, Tyr, Val, Phe, Leu, or Ile;
- and Xaa.sub.10 is any amino acid other than Asp or Glu, and wherein said biotinylation-peptide is capable of being biotinylated by a biotin ligase at said lysine residue adjacent to Xaa.sub.6 ;
- and is 13 to 50 amino acids in length;
- (b) producing said fusion protein encoded by said vector either by transforming a recombinant host cell with said vector and culturing host cells transformed with the vector or by incubating said vector in a cell-free transcription and translation system;
- and
- (c) incubating said fusion protein in a reaction mixture comprising biotin or a biotin analogue and a biotinylation enzyme, resulting in biotinylation of said fusion protein.
12 more elements
A recombinant DNA vector that comprises a nucleic acid that encodes a biotinylation peptide wherein said biotinylation peptide comprises an amino acid sequence defined by:
- Leu Xaa.sub.1 Xaa.sub.2 Ile Xaa.sub.3 Xaa.sub.4 Xaa.sub.5 Xaa.sub.6 Lys Xaa.sub.7 Xaa.sub.8 Xaa.sub.9 Xaa.sub.10 (SEQ ID NO:1), where Xaa.sub.1 is any amino acid;
- Xaa.sub.2 is any amino acid other than Leu, Val, Ile, Trp, Phe, or Tyr;
- Xaa.sub.3 is Phe or Leu;
- Xaa.sub.4 is Glu or Asp;
- Xaa.sub.5 is Ala, Gly, Ser, or Thr;
- Xaa.sub.6 is Gln or Met;
- Xaa.sub.7 is Ile, Met, or Val;
- Xaa.sub.8 is Glu, Leu, Val, Tyr, or Ile;
- Xaa.sub.9 is Trp, Tyr, Val, Phe, Leu, or Ile;
- and Xaa.sub.10 is any amino acid other than Asp or Glu, and wherein said biotinylation-peptide is capable of being biotinylated by a biotin ligase at said lysine residue adjacent to Xaa6;
- and is 13 to 50 amino acids in length.
8 more elements
A kit for biotinylating a protein, the kit comprising a recombinant DNA expression polynucleotide that encodes a biotinylation peptide peptide wherein said biotinylation peptide comprises an amino acid sequence defined by:
- Leu Xaa.sub.1 Xaa.sub.2 Ile Xaa.sub.3 Xaa.sub.4 Xaa.sub.5 Xaa.sub.6 Lys Xaa.sub.7 Xaa.sub.8 Xaa.sub.9 Xaa.sub.10 (SEQ ID NO:1), where Xaa.sub.1 is any amino acid;
- Xaa.sub.2 is any amino acid other than Leu, Val, Ile, Trp, Phe, or Tyr;
- Xaa.sub.3 is Phe or Leu;
- Xaa.sub.4 is Glu or Asp;
- Xaa.sub.5 is Ala, Gly, Ser, or Thr;
- Xaa.sub.6 is Gln or Met;
- Xaa.sub.7 is Ile, Met, or Val;
- Xaa.sub.8 is Glu, Leu, Val, Tyr, or Ile;
- Xaa.sub.9 is Trp, Tyr, Val, Phe, Leu, or Ile;
- and Xaa.sub.10 is any amino acid other than Asp or Glu;
- wherein said biotinylation-peptide is capable of being biotinylated by a biotin ligase at said lysine residue adjacent to Xaa.sub.6 ;
- and is 13 to 50 amino acids in length and wherein said biotinylation protein can be fused in frame with a protein by inserting the coding sequence for the protein.
9 more elements
Dependencies are inferred from the claim text.