Large scale expression and purification of mouse melanopsin-L in the baculovirus expression system.
Melanopsin is the mammalian photopigment that primarily mediates non-visual photoregulated physiology. So far, this photopigment is poorly characterized with respect to structure and function. Here, we report large-scale production and purification of the intact long isoform of mouse melanopsin (melanopsin-L) using the baculovirus/insect cell expression system. Exploiting the baculoviral GP67 signal peptide, we obtained expression levels that varied between 10-30pmol/10(6)cells, equivalent to 2-5mg/L. This could be further enhanced using DMSO as a chemical chaperone. LC-MS analysis confirmed that full-length melanopsin-L was expressed and demonstrated that the majority of the expressed protein was N-glycosylated at Asn(30) and Asn(34). Other posttranslational modifications were not yet detected. Purification was achieved exploiting a C-terminal deca-histag, realizing a purification factor of several hundred-fold. The final recovery of purified melanopsin-L averaged 2.5% of the starting material. This was mainly due to low extraction yields, probably since most of the protein was present as the apoprotein. The spectral data we obtained agree with an absorbance maximum in the 460-500nm wavelength region and a significant red-shift upon illumination. This is the first report on expression and purification of full length melanopsin-L at a scale that can easily be further amplified.
Department of Biochemistry, Radboud University Nijmegen Medical Centre, Nijmegen Centre for Molecular Life Sciences, P.O. Box 9101, NL-6500 HB Nijmegen, The Netherlands. Electronic address: nazhatshirzad@gmail.com.
Nazhat
N
Shirzad-Wasei
Jenny
J
van Oostrum
Petra H
PH
Bovee-Geurts
Maud
M
Wasserman
Giel J
GJ
Bosman
Willem J
WJ
Degrip
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D003902:Detergents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D017299:Rod Opsins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D061987:Sf9 Cells; D012995:Solubility; D000099210:Melanopsin
D003902:Detergents; D011994:Recombinant Proteins; D017299:Rod Opsins; D000099210:Melanopsin
Baculovirus expression system; Chaperones; Melanopsin-L; Posttranslational modification; Purification; Spectral properties