Expression, purification, crystallization and diffraction analysis of a selenomethionyl lipase Lip8 from Yarrowia lipolytica.

Yarrowia lipolytica is a non-conventional model microorganism with multiple biotechnological applications. It is also considered to be an excellent producer for lipase. Genome survey shows that Y. lipolytica possesses various paralogs of genes coding for extracellular, cell-bound and intracellular lipolytic enzymes. However, little structural information on these isoenzymes is available. With the aim to facilitate crystal structure solution of Lip8, one of the most valuable lipases from Y. lipolytica, a less conventional protein expression technique-selenomethionyl protein expression (SPE) was employed to produce recombinant SeMet-Lip8 in Escherichia coli. At last, three Met residues of Lip8 were all substituted with SeMet. A total of 72 mg of SeMet-Lip8 were obtained from a liter of the SeMet medium. Using sodium acetate as a precipitant and ammonium sulfate as an additive, crystals of the SeMet-Lip8 with 1.9 Å were sucessfully cultured through hanging-drop vapour-diffusion method. The estimated crystal dimensions were 0.11 × 0.11 × 0.14 mm. The crystal belonged to the space group I4 with unit cell parameters a = b = 128.87 Å, c = 171.77 Å, α = β = γ = 90˚. It is the second member of lipase crystal family from Y. lipolytica. This work will provide a platform for further studying lipases from a structural insight.

Citation

Jun Sheng, XiaoFeng Ji, Yuan Zheng, Mi Sun. Expression, purification, crystallization and diffraction analysis of a selenomethionyl lipase Lip8 from Yarrowia lipolytica. Preparative biochemistry & biotechnology. 2016 Jul 5

PMID: 27380164

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