Expression and identification of human LIGHT-Fc fusion protein in Pichia pastoris

Objective To prepare human LIGHT-Fc fusion protein by using yeast expression system. Methods The recombinant plasmid pPIC9K-I.IGHT-Fc containing the gene of outer membrane domain of human LIGHT and human IgG4 Fc was constructed by genetic engineering technology, linearized with Sal I and electrotransformed to competent yeast GS115. The recombinant transformants were identified by PCR, of which the positive ones were induced with methanol. The transcription level of target gene was determined by RT-PCR, while the expressed product was identified by SDS-PAGE and Western blot. Results Restriction analysis and sequencing proved that recombinant plasmid pPIC9K-LIGHT-Fc was constructed correctly. All the ten recombinants were Mut⁺ transformants, from which a specific target gene fragment was amplified after induction with methanol. The expressed fusion protein LIGHT-Fc, with a relative molecular mass of about 45 000, showed specific binding to mouse anti-human LIGHT polyclonal antibody. Conclusion Fusion protein LIGHT-Fc was expressed successfully in P. pastoris, which laid a foundation of further study on its biological function.