Expression, purification, and characterization of minimized chicken riboflavin carrier protein from a synthetic gene in Escherichia coli.
Minimized proteins have long been used to elicit immune response to particular regions of a protein antigen. Most efforts to derive minimized proteins have employed synthetic peptide fragments. Here we describe molecular cloning and production of a minimized chicken riboflavin carrier protein (mini-RCP) sequence that harbours all the four neutralizing epitopes but lacks the sequences that otherwise elicit undesirable antibodies. The gene encoding mini-RCP is engineered by contiguous alignment of nucleotide sequences coding for selected epitopes of chicken RCP separated by leucyl alanine residues. The gene has been constructed from eight oligonucleotides by employing overlapping PCR strategy and expressed in Escherichia coli, using the T7 promoter system. The recombinant protein could be purified to homogeneity by a single step Ni2+ affinity chromatography. Western blot experiments using epitope specific antisera confirm that the corresponding linear amino acid sequences are available for immunorecognition in the engineered protein. This methodology enables continuous production and purification in bulk amounts of the minimized RCP as a source of candidate immunocontraceptive vaccine in mammals.
Subramanian
Sarada
S
Department of Molecular Reproduction Development and Genetics, Indian Institute of Science, 560 012, Bangalore, India. sarada@mrdg.iisc.ernet.in
Kondaiah
Paturu
P
Radhakantha Adiga
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data
D002352:Carrier Proteins; D026901:Membrane Transport Proteins; C019079:riboflavin-binding protein