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Production of recombinant endotoxin neutralizing protein in Pichia pastoris and methods for its puriﬁcation
Production of recombinant Limulus endotoxin neutralizing protein (rENP) was attained with the GS115 methylotrophic strain ofPichia pastoris transformed with a plasmid, bearing multiple ENP gene copies.The synthetic gene for Limulus ENP was cloned into the integrative plasmid pAO815 under the control of a methanol-inducible promoter. Clones containing a single enp insert were used to construct cassettes bearing 2 and 3 tandem copies of enp. These were then integrated at the HIS locus of P. pastoris GS115 (his4).Clones were chosen for their ability to produce rENP upon methanol induction in shaker ﬂasks, and then the 1x, 2x, and 3x-enp strains were analyzed by Southern blot for the presence of the ENP gene(s). Isolate 3x5q, containing a 3x-enp cassette, was the best producer of rENP. Under optimal conditions this strain grown in a fed-batch mode produced yields of >500mg rENP/Lwith an average of 5.46mg rENP/g DCW. Puriﬁcation of rENP from the clariﬁed broth resulted in a yield of 35% and a purity of >86%.
Glycosylated rENP, the main contaminant, was removed with a concanavalin-A column and characterized. The pure rENP neutralized lipopolysaccharide and had the mass, amino-acid composition and N-terminal sequence expected from the cloned gene.2002 Elsevier Science (USA). All rights reserved.

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