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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)
 
 
 

Biochemical, biophysical, and pharmacological characterization of bacterially expressed human agouti-related protein.

The agouti-related protein gene ( Agrp) is a novel gene implicated in the control of feeding behavior. The hypothalamic expression of Agrp is regulated by leptin, and overexpression of Agrp in transgenic animals results in obesity and diabetes. By analogy with the known actions of agouti, these data suggest a role for the Agrp gene product in the regulation of melanocortin receptors expressed in the central nervous system. The availability of recombinant, highly purified protein is required to fully address this potential interaction. A nearly full-length form of AGRP (MKd5-AGRP) was expressed in the cytosolic or soluble fraction of Escherichia coli and appeared as large intermolecular disulfide-bonded aggregates. Following oxidation, refolding, and purification, this protein was soluble, and eluted as a single symmetric peak on RP-HPLC. Circular dichroism studies indicated that the purified protein contains primarily random coil and beta-sheet secondary structure. Sedimentation velocity studies at neutral pH demonstrated that MKd5-AGRP is monomeric at low micromolar concentrations. Mobility shifts observed using SDS-PAGE under reducing and nonreducing conditions for bacterially expressed and mammalian expressed AGRP were identical, an indication of a similar disulfide structure. The purification to homogeneity of a second, truncated form of AGRP (Md65-AGRP) which was expressed in the insoluble or inclusion body fraction is also described. Both forms act as competitive antagonists of alpha-melanocyte stimulating hormone (alpha-MSH) at melanocortin-3 (MC-3) and melanocortin-4 receptors (MC-4). The demonstration that AGRP is an endogenous antagonist with respect to these receptors is a unique mechanism within the central nervous system, and has important implications in the control of feeding.[1]

References

  1. Biochemical, biophysical, and pharmacological characterization of bacterially expressed human agouti-related protein. Rosenfeld, R.D., Zeni, L., Welcher, A.A., Narhi, L.O., Hale, C., Marasco, J., Delaney, J., Gleason, T., Philo, J.S., Katta, V., Hui, J., Baumgartner, J., Graham, M., Stark, K.L., Karbon, W. Biochemistry (1998) [Pubmed]
 
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