Abstract

GOMEZ OSORIO, Valentina et al. Obtaining the recombinant green fluorescent protein and its IgY polyclonal antibody. Rev. colomb. biotecnol [online]. 2023, vol.25, n.1, pp.57-68.  Epub Dec 11, 2023. ISSN 0123-3475.  https://doi.org/10.15446/rev.colomb.biote.v25n1.91675.

Green Fluorescent Protein (GFP) is widely used in in vivo and in vitro assays. Multiple variants of this protein have been generated to diversify its characteristics, such as the enhancer GFP (EGFP) that emits a 35-fold higher fluorescence signal compared to the wild-type protein, being implemented as a fusion reporter in localization and structural stability studies, among others. Detection of this protein can be direct or indirect, fusing anti-GFP antibodies. Although the use of GFP is generalized and of evident utility in research and teaching, the molecular tools for its study exhibit a high cost since they must be imported, constituting a limited resource in Colombia. This work reports the cloning and expression of the recombinant protein 6xHisEGFP, which purification was completed from the soluble and insoluble fraction of the heterologous Escherichia coli system by immobilized metal affinity chromatography and preparative SDS-PAGE, respectively. The purified protein was implemented as an antigen to produce avian polyclonal antibodies (IgY) against EGFP, which were obtained from collected eggs and blood serum from immunized hens. In this sense, the proposed methodological strategy constitutes an advance in the development of a biotechnological system for the national production of molecular tools such as avian polyclonal antibodies at low-cost.

Keywords : Biotechnology; EGFP; Recombinant proteins.

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