Endocrine Abstracts

Introduction: Anosmin-1, which is implicated in the pathogenesis of X-linked Kallmann`s syndrome, consists of a cysteine rich region, followed by a WAP domain and 4 fibronectin type III (FN3) domains. The N-terminal region of anosmin-1, WAP and 1st FN3 domain, is highly conserved in a wide variety of species which suggests that the activity of the protein resides in these conserved segments. Difficulties in the expression of the full-length anosmin-1 by Chinese hamster ovary cells and deficiency of post-translational modification by E.coli, have resulted in a limited set of functional studies. To find an effective and quick system, we used insect cells -Drosophila S2 cells- and Ni-NTA agarose chromatography to generate histidine-tagged full-length and truncated anosmin-1 including N-terminal WAP and 1st FN3.

Methods: The whole KAL1 cDNA (PIWF4) and the fragment (PIWF1) encoding the N-terminal 289 aa residues were cloned into the expression vector pMT/BiP/V5-His and co-transfected with selection vector pCoHYGRO into S2 cells. The stable cells were selected with 300mg/ml and 700mg/ml hygromycin B and induced in either 300cm² flasks or shake flasks. The conditioned medium and 350mM NaCl extraction were collected , mixed with Ni-NTA agarose at the ratio of 1: 20 for 1.5 hours. The protein was eluted by 250mM imidazole.

Results: Compared with 300mg/ml hygromycin selection, the 700mg/ml hygromycin- selected cells produced 10 fold more protein (about 100mg/10⁹cells of PIWF4 and 560mg/10⁹cells of PIWF1). In addition, the expression efficiency with static flasks was about 40 fold higher than that with shake flasks.

Discussion and conclusions:

Drosophila S2 cells and Ni-NTA agarose chromatography for pure histidine-tagged anosmin-1 is an effective, simple and quick method of protein production. Expression was highest using 700mg/ml hygromycin selection when cells grown in a static flask . Biochemically pure protein will enable functional and structural assays studies for anosmin-1.