Expression, purification and characterization of leukotriene B(4) receptor, BLT1 in Pichia pastoris.
The high yield expression of BLT1, a G-protein coupled receptor for leukotriene B(4), was established in Pichia pastoris for structural studies. Guinea pig BLT1 was expressed in a functional form without post-translational modifications for the rapid purification and the crystallization. Among the BLT1s from four species, only guinea pig BLT1 was successfully expressed with the comparable binding affinity to BLT1 of native guinea pig tissues for several ligands. Only Asn4 of the two putative N-glycosylation sites was glycosylated, and the mutation to Ala to avoid glycosylation did not affect the ligand binding affinity. However, the N-terminal region of the mutant was digested at the carboxyl ends of Arg3 and Arg8, as detected by N-terminal amino acid sequencing, and Ser309 in the C-terminal region was partially phosphorylated, as identified in the micro-sequencing by Q-TOF-MS/MS. To avoid chemical heterogeneity, the N-terminal peptide (1-14) truncated and the C-terminal phosphorylation-site eliminated mutant was generated. The binding affinity of the mutant's membrane fraction for LTB(4) was K(d)=6.6 nM and B(max)=50.0 pmol/mg membrane protein. The yield of purified mutant was approximately 0.3-0.4 mg from 1L culture, and the protein showed a single peak at molecular weight of 100 kDa in gel-filtration and no glycosylation or phosphorylation in MALDI-TOF MS.
Hori
Tetsuya
T
Structural Biophysics Laboratory, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
Sato
Yo
Y
Takahashi
Naoko
N
Takio
Koji
K
Yokomizo
Takehiko
T
Nakamura
Motonao
M
Shimizu
Takao
T
Miyano
Masashi
M
D000595:Amino Acid Sequence; D000818:Animals; D015870:Gene Expression; D006031:Glycosylation; D006168:Guinea Pigs; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D010843:Pichia / Q000235:genetics*; D011485:Protein Binding; D018102:Receptors, Leukotriene B4 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D015854:Up-Regulation
D018102:Receptors, Leukotriene B4