High-level expression, purification, and crystallization of recombinant rat leukotriene C(4) synthase from the yeast Pichia pastoris.
Leukotriene C(4) synthase (LTC4S) is a member of the MAPEG family of integral membrane proteins and catalyzes the conjugation of leukotriene A(4) with glutathione to form leukotriene C(4), a powerful mediator of allergic inflammation and anaphylaxis. Structural information on this class of proteins would be highly useful for rational drug design. Here, we report the expression, purification, and crystallization of recombinant LTC4S from rat. The enzyme was expressed as an N-terminal hexa-histidine-tagged fusion protein in Pichia pastoris and purified with two steps of affinity chromatography on Ni-Sepharose and S-hexyl-glutathione agarose, followed by gel filtration. From 1l culture, we obtained 0.5-1 mg of apparently homogeneous protein with a specific LTC4S activity ranging between 36 and 49 micromol/mg/min. A small-scale screen identified dodecyl maltoside as a useful detergent for protein extraction and yielded a highly active protein. When tested separately in crystallization trials of the purified LTC4S, six out of seven detergents from all the maltoside family yielded diffracting crystals with the highest resolution at approximately 6 A. Hence, our approach holds promise for solving the structure of rat LTC4S and other members of the MAPEG family of integral membrane proteins.
Wetterholm
Anders
A
Department of Medical Biochemistry and Biophysics, Division for Chemistry 2, Karolinska Institutet, S-171 77 Stockholm, Sweden.
Martinez Molina
Daniel
D
Nordlund
Pär
P
Eshaghi
Said
S
Haeggström
Jesper Z
JZ
D000818:Animals; D003460:Crystallization; D005982:Glutathione Transferase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010843:Pichia / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D005982:Glutathione Transferase; C041116:leukotriene-C4 synthase