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 w as 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 h ybridizing clone (AqpBF1), contained an insert of 1878 bp, and DNA sequence analysis revealed an open reading frame of 753 bp encoding a polypeptide o f predicted M-r = 26 163 Da, Comparison of the AqpBF1 deduced protein seque nce with the GenBank database revealed significant homology to many aquapor in genes, including 72% identity with a partial DNA sequence encoding a mem ber (DRIP) Of the MIP protein family isolated from Drosophila melanogaster. The most closely related, full-length, GenBank sequence was an aquaporin g ene isolated from the digestive tract of the sap-sucking insect Cicadella v iridis, which was 53% identical to the buffalo fly AqpBF1 protein sequence. The full-length coding sequence of AqpBF1 was cloned into the (His)(6)-fus ion 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 protei n was solubilized from E. coli membranes with n-octyl beta-D-glucopyranosid e and purified by affinity chromatography on a Ni++-sepharose column in the presence of detergent.