Expression, purification, and characterization of minimized chicken riboflavin carrier protein from a synthetic gene in Escherichia coli

Subramanian, Sarada and Kondaiah, Paturu and Adiga, Radhakantha P (2002) Expression, purification, and characterization of minimized chicken riboflavin carrier protein from a synthetic gene in Escherichia coli. In: Protein Expression and Purification, 26 (2). pp. 284-289.

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Abstract

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 $Ni^{2+}$ 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.

Item Type:	Journal Article
Publication:	Protein Expression and Purification
Publisher:	Elsevier Science
Additional Information:	Copyright of this article belongs to Elsevier Science.
Keywords:	Riboflavin carrier protein;Minimized protein;Synthetic gene;Overlapping PCR;Hexahistidine tag
Department/Centre:	Division of Biological Sciences > Molecular Reproduction, Development & Genetics
Date Deposited:	16 Oct 2007
Last Modified:	01 Mar 2012 06:50
URI:	http://eprints.iisc.ac.in/id/eprint/12292

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