Cloning, expression and purification of the fire ant chemosensory protein 1 in the yeast Kluyveromyces lactis
Previously, the fire ant (Solenopsis invicta) antenna-specific chemosensory protein-gene 1 (CSP1) was cloned (Gonzalez D. et al. 2009). In this study the gene was directionally inserted into the Kluyveromyces lactis expression vector pKLAC1 using pre-existing XhoI and PacI restriction sites. This was done by first mutagenizing the gene to create the two restriction sites, XhoI and PacI. Then the CSP1 gene was cloned into the pCR®-Blunt vector (InvitrogenTM), by cutting out the gene with XhoI and PacI, and then introducing the gene into pKLAC1. This new vector, pKLAC1/CSP1, was cloned into E. coli TOP 10 cells to screen and propagate the vector and then the linearized vector was transformed into the yeast Kluyveromyces lactis by chemical transformation to ultimately express the CSP1 protein.
By using the plasmid to transform our gene into the yeast, we found one disadvantage in the method. The K. lactis protein expression kit from New England Biolabs® does not provide a clear method to discriminate between transformed or untransformed colonies. Therefore, we have attempted to introduce the green florescent protein (GFP) into the plasmid by mutagenizing the pKLAC1 to create more restriction sites and use those sites to clone in the GFP. We cloned the GFP fragment into pCR®-Blunt vector and by cutting with NotI, it is then introduced into pKLAC1, now called pKLAC1/GFP.