Molecular Cloning And Expression In Escherichia Coli Of An Aquaporin-Like Gene From Adult Buffalo Fly (Haematobia Irritans Exigua)

A gene fragment encoding a putative member of the aquaporin gene family was amplified using cDNA prepared from unfed adult buffalo fly poly(A)(+) RNA and degenerate PCR primers designed from highly conserved regions of amino acids found in ail members of the aquaporin gene family. This PCR product was labelled with digoxigenin-dUTP and used as a probe to screen a lambda gt-11 cDNA library constructed from unfed adult buffalo fly. One positively hybridizing clone (AqpBF1), contained an insert of 1878 bp, and DNA sequence analysis revealed an open reading frame of 753 bp encoding a polypeptide of predicted M-r = 26 163 Da, Comparison of the AqpBF1 deduced protein sequence with the GenBank database revealed significant homology to many aquaporin genes, including 72% identity with a partial DNA sequence encoding a member (DRIP) Of the MIP protein family isolated from Drosophila melanogaster. The most closely related, full-length, GenBank sequence was an aquaporin gene isolated from the digestive tract of the sap-sucking insect Cicadella viridis, which was 53% identical to the buffalo fly AqpBF1 protein sequence. The full-length coding sequence of AqpBF1 was cloned into the (His)(6)-fusion vector, pQE10, and the recombinant protein was expressed in Escherichia coli following induction by IPTG. The recombinant (His)(6)-fusion protein was localized predominantly in the membrane fraction of E, coli. The protein was solubilized from E. coli membranes with n-octyl beta-D-glucopyranoside and purified by affinity chromatography on a Ni++-sepharose column in the presence of detergent.