Cm. Elvin et al., Molecular cloning and expression in Escherichia coli of an aquaporin-like gene from adult buffalo fly (Haematobia irritans exigua), INSEC MOL B, 8(3), 1999, pp. 369-380
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.