title: Functional expression of porcine aminoacylase 1 in E. coli using a codon optimized synthetic gene and molecular chaperones.
abstract: Efficient recombinant expression of N-acyl-L-aminoacylase 1 from pig kidney (pAcy1) was achieved in the prokaryotic host Escherichia coli. An optimized nucleotide sequence (codon adaptation index 0.95 for E. coli), was cloned into vector pET-52(b) yielding an E. coli-expressible pAcy1 gene. Formation of inclusion bodies was alleviated by co-expression of molecular chaperones resulting in 2.7- and 4.2-fold increased recovery of active pAcy1 using trigger factor or GroEL-GroES, respectively. Facile purification was achieved via StrepTag affinity chromatography. Overall, more than 80 mg highly active pAcy1 (94 U/mg) was obtained per liter of cultivation broth. The protein was analyzed for structural and functional identity, and the performances of further described expression and purification systems for pAcy1 were compared.
authors.affiliation: Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Felix-Hausdorff-Str. 4, D-17487, Greifswald, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Wardenga
authors.forename: Frank
authors.initials: F
authors.lastname: Hollmann
authors.forename: Oliver
authors.initials: O
authors.lastname: Thum
authors.forename: Uwe
authors.initials: U
authors.lastname: Bornscheuer
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D003001:Cloning, Molecular; D003062:Codon; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005813:Genes, Synthetic; D005822:Genetic Vectors / Q000235:genetics; D018832:Molecular Chaperones / Q000235:genetics* / Q000378:metabolism; D008970:Molecular Weight; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013552:Swine
chemical_list: D003062:Codon; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D000581:Amidohydrolases; C019672:aminoacylase I