例如:"lncRNA", "apoptosis", "WRKY"

Constitutive expression of human gastric lipase in Pichia pastoris and site-directed mutagenesis of key lid-stabilizing residues.

Biochim. Biophys. Acta. 2017 Oct;1862(10 Pt A):1025-1034. Epub 2017 Jul 08
Laura Sams 1 , Sawsan Amara 2 , Almahdi Chakroun 3 , Sébastien Coudre 3 , Julie Paume 4 , Jacqueline Giallo 4 , Frédéric Carrière 5
Laura Sams 1 , Sawsan Amara 2 , Almahdi Chakroun 3 , Sébastien Coudre 3 , Julie Paume 4 , Jacqueline Giallo 4 , Frédéric Carrière 5
+ et al

[No authors listed]

Author information
  • 1 CNRS, Aix Marseille Université, Enzymologie Interfaciale et Physiologie de la Lipolyse UMR7282, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France; GERME S.A., Technopôle Marseille Provence Château-Gombert, ZAC la Baronne, 12 Rue Marc Donadille, 13013 Marseille, France.
  • 2 CNRS, Aix Marseille Université, Enzymologie Interfaciale et Physiologie de la Lipolyse UMR7282, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France; Lipolytech, Zone Luminy Biotech, 163 avenue de Luminy, 13288 Marseille Cedex 09, France.
  • 3 CNRS, Aix Marseille Université, Enzymologie Interfaciale et Physiologie de la Lipolyse UMR7282, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
  • 4 GERME S.A., Technopôle Marseille Provence Château-Gombert, ZAC la Baronne, 12 Rue Marc Donadille, 13013 Marseille, France.
  • 5 CNRS, Aix Marseille Université, Enzymologie Interfaciale et Physiologie de la Lipolyse UMR7282, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France. Electronic address: carriere@imm.cnrs.fr.

摘要


The cDNA encoding human gastric lipase (HGL) was integrated into the genome of Pichia pastoris using the pGAPZα A transfer vector. The HGL signal peptide was replaced by the yeast α-factor to achieve an efficient secretion. Active rHGL was produced by the transformed yeast but its levels and stability were dependent on the pH. The highest activity was obtained upon buffering the culture medium at pH5, a condition that allowed preserving enzyme activity over time. A large fraction (72±2%) of secreted rHGL remained however bound to the yeast cells, and was released by washing the cell pellet with an acid glycine-HCl buffer (pH2.2). This procedure allowed establishing a first step of purification that was completed by size exclusion chromatography. N-terminal sequencing and MALDI-ToF mass spectrometry revealed that rHGL was produced in its mature form, with a global mass of 50,837±32Da corresponding to a N-glycosylated form of HGL polypeptide (43,193Da). rHGL activity was characterized as a function of pH, various substrates and in the presence of bile salts and pepsin, and was found similar to native HGL, except for slight changes in pH optima. We then studied by site-directed mutagenesis the role of three key residues (K4, E225, R229) involved in salt bridges stabilizing the lid domain that controls the access to the active site and is part of the interfacial recognition site. Their substitution has an impact on the pH-dependent activity of rHGL and its relative activities on medium and long chain triglycerides.

KEYWORDS: Acid lipase, Heterologous protein expression, Lipid digestion, Lipolysis, Yeast