title: Soluble variants of human recombinant glutaminyl cyclase.
abstract: Recombinant human Glutaminyl Cyclase expressed in E. coli is produced as inclusion bodies. Lack of glycosylation is the main origin of its accumulation in insoluble aggregates. Mutation of single isolated hydrophobic amino acids into negative amino acids was not able to circumvent inclusion bodies formation. On the contrary, substitution with carboxyl-terminal residues of two or three aromatic residues belonging to extended hydrophobic patches on the protein surface provided soluble but still active forms of the protein. These mutants could be expressed in isotopically enriched forms for NMR studies and the maximal attainable concentration was sufficient for the acquisition of (1)H-(15)N HSQC spectra that represent the starting point for future drug development projects targeting Alzheimer's disease.
authors: Pharmacology Department, Siena Biotech, Siena, Italy.
authors: Cristiana
authors: C
authors: Castaldo
authors: Silvia
authors: S
authors: Ciambellotti
authors: Raquel
authors: R
authors: de Pablo-Latorre
authors: Daniela
authors: D
authors: Lalli
authors: Valentina
authors: V
authors: Porcari
authors: Paola
authors: P
authors: Turano
mesh_terms: D019881:Aminoacyltransferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D055672:Static Electricity
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; D019881:Aminoacyltransferases; C059280:glutaminyl-peptide cyclotransferase
references: Proc Natl Acad Sci U S A. 2005 Sep 13;102(37):13117-22
references: 16135565
references: J Biol Chem. 1988 Jun 25;263(18):8832-7
references: 3288624
references: Nat Struct Biol. 2002 Oct;9(10):729-33
references: 12219082
references: Proc Natl Acad Sci U S A. 1987 Jun;84(11):3628-32
references: 3473473
references: Curr Opin Biotechnol. 1999 Oct;10(5):411-21
references: 10508629
references: J Pharm Sci. 2005 Aug;94(8):1626-35
references: 15959882
references: Nat Protoc. 2010 May;5(5):883-97
references: 20431534
references: J Mol Biol. 2010 Aug 20;401(3):374-88
references: 20558177
references: J Biol Chem. 2005 Oct 28;280(43):35815-21
references: 16105836
references: Biochim Biophys Acta. 1999 Jul 13;1432(2):214-21
references: 10407143
references: Protein Expr Purif. 2005 Sep;43(1):65-72
references: 16084398
references: J Biol Chem. 2011 Apr 8;286(14):12439-49
references: 21288892
references: Biochemistry. 2002 Sep 3;41(35):10849-57
references: 12196024
references: Biochem J. 2008 Apr 1;411(1):181-90
references: 18072935
references: Biochemistry. 2011 Jul 19;50(28):6280-8
references: 21671571
references: Nat Med. 2008 Oct;14(10):1106-11
references: 18836460
references: Biochemistry. 2002 Jan 8;41(1):1-7
references: 11771996
references: Anal Biochem. 2002 Apr 1;303(1):49-56
references: 11906150
references: Angew Chem Int Ed Engl. 2003 Feb 24;42(8):864-90
references: 12596167
references: Nat Rev Drug Discov. 2008 Sep;7(9):738-45
references: 19172689
references: Biochem Biophys Res Commun. 2012 Oct 26;427(3):677-81
references: 23036197
references: J Med Chem. 2006 Jan 26;49(2):664-77
references: 16420052
references: J Am Chem Soc. 2004 Jul 28;126(29):8933-9
references: 15264823
references: J Am Chem Soc. 2003 Feb 19;125(7):1731-7
references: 12580598
references: J Biol Chem. 2003 Dec 12;278(50):49773-9
references: 14522962