Endocrine Abstracts

Introduction: RAMPs (receptor activity-modifying proteins) are single-pass transmembrane accessory proteins required for appropriate signalling of the G-protein coupled receptors; CRLR (Calcitonin receptor like receptor) and CTR (Calcitonin receptor). There are three forms of human RAMPs known as, RAMP1, 2 and 3. RAMP3 heterodimerises with CRLR or CTR to form a high affinity receptor for the peptide hormones adrenomedullin and amylin, respectively. However, the exact mechanism by which RAMP3 interacts with these receptors remains unclear. Our aim was the purification of the RAMP3 extracellular domain in order to undertake structural studies.

Method: RAMP3 was expressed in both E. coli and mammalian cells. For E. coli expression the DNA sequence encoding the extracellular domain of RAMP3 was linked to N-terminal 6 Histidine tag and cloned into the bacterial expression plasmid pET21(a+). The protein was then expressed in the bacterial strain BL21 (DE3)-RIPL cell using TB medium. For mammalian expression stable clones were made in CHO cells under a CMV promoter.

Results: In E. coli significant expression level of RAMP3 (ECD) was seen but most of it was present as inclusion bodies. Inclusion bodies were isolated, denatured and renatured in the presence of arginine. The refolded protein was purified using Nickel affinity columns. Inclusion bodies solubilised well in 6M Guanidine, however a significant fraction of the purified protein showed tendency to form aggregates after refolding. Mammalian expression resulted in soluble RAMP3 which was highly glycosylated and initial purification revealed a protein of about 250 kDa.

Conclusion: The E. coli derived RAMP3 (ECD) tends to form aggregates after being refolded from inclusion bodies and this is probably due to intermolecular disulfide bonds or because the protein is not glycosalated. Mammalian expression gave soluble RAMP3 which was highly glycosylated. The future challenge will be to purify RAMP3 at sufficient concentration that is stable and uniform.