Cloning, expression and purification of fire ant chemosensory protein 3 and odorant binding protein 1 in the yeast Kluyveromyces lactis.

摘要

A shotgun proteomic analysis of the fire ant (Solenopsis invicta ) antenna showed the expression of two chemosensory proteins, CSP1 and CSP3 (Gonzalez D. et al. 2009). This suggested that CSP3 may have a role in nestmate recognition. The function of CSP3 could be tested by heterologous expression. In this study the coding sequence for CSP3 was directionally inserted into the Kluyveromyces lactis expression vector pKLAC1 using pre-existing XhoI and PacI restriction sites. This new vector, pKLAC1/CSP3, was linearized and then chemically transformed into the yeast K. lactis to express CSP3 protein. The expressed CSP3 was secreted into the media then purified using a HIC column and ammonium sulfate precipitation. Ligand binding studies were carried out by adding N-phenyl-1-naphthylamine (a fluorescent dye) to CSP3, which showed that ligand was binding cooperative with a dissociation constant of 13.4 microM, and a Hill constant of 1.45.;The previous proteomic analysis (Gonzalez D. et al. 2009) also showed an odorant binding protein1 (OBP1) expressed in fire ant antennae. OBP1 has a high degree of amino acid sequence similarity to honey bee OBP1, which is known to carry signaling molecules released from the queen's mandibular gland. To understand the role of fire ant OBP1 we have also endeavored to express it. The coding sequence for OBP1 was introduced into pKLAC1 and transformed in K. lactis. The protein product was smaller product than expected, possibly due to the presence of a cryptic intron. Site directed mutagenesis was performed to remove suspected intron sites failed to produce a full length OBP1. To overcome this we constructed a codon optimized E. coli gene, introduced it into pET21a and electroporated into E. coli BL 21 cells. However, this did not express OBP1. As a last ditch effort, eOBP1 gene was introduced into the pKLAC1 vector and transformed into K. lactis. This expressed a smaller protein product as earlier (native and cryptic OBP1). As eOBP1 was a completely different gene and the DNA sequence also does not contain any possible cryptic intron sites. There might be any anomalous migration problem in SDS-PAGE gel and recently full length protein sample was sent for TOF analysis.