ABSTRACT
In the title mol-ecule, C22H22N2O4, the eth-oxy-phenyl rings are oriented at dihedral angles of 69.31â (13) and 75.90â (13)° to the nitro-phenyl ring and are twisted to each other, making a dihedral angle of 78.55â (13)°. In the crystal, weak C-Hâ¯O hydrogen bonds and C-Hâ¯π inter-action link the mol-ecules into a three-dimensional supra-molecular architecture.
ABSTRACT
The mouse metallothionein (mMT) mutant alpha-KKS-alpha has a higher capacity for binding heavy metals than wild type mMT. The mMT mutant alpha-KKS-alpha gene was placed under the control of the strong promoter PpbsA to generate the intermediate vector pRL-alpha-KKS-alpha. pRLalpha-KKS-alpha was then linked with the plasmid pDC-08 to construct shuttle expression vector pDC-alphaKKS-alpha. This expression vector was transformed into Anabaena sp. PCC 7120 using triparental conjugative transfer. After antibiotic selection (ampicillin and kanamycin), transgenic Anabaena was identified by PCR and Western blotting. The expression level of the mMT mutation alpha-KKS-alpha reached 7.4 mg/g dry cells weight, as detected by ELISA, and heavy metal resistance of the transgenic Anabaena was significantly improved.
Subject(s)
Anabaena/genetics , Gene Expression Regulation , Metallothionein/biosynthesis , Metallothionein/genetics , Metals, Heavy/pharmacology , Anabaena/physiology , Blotting, Western , Cloning, Molecular , Drug Resistance , Enzyme-Linked Immunosorbent Assay , Plasmids , Polymerase Chain ReactionABSTRACT
To express BACE (beta-site APP cleaving enzyme), an aspartic protease related to Alzheimer's disease in E.coli to obtain the active protein after refolding and purification, the sequence for soluble form of BACE was inserted into prokaryotic expression vector pET11a. After expression in E.coli BL21(DE3), the protein was obtained as inclusion bodies, after refolding and purification. The proteolytic activity of the protein was measured by HPLC and MS. After refolding and purification, the protein exhibited beta-secretase activity by cleaving the synthetic peptide, which was designed according to the beta-secretase site at Swedish mutant of APP, and according Hanes method the kinetics constants for the synthetic peptide of BACE protein were measured. The study will facilitate BACE protein structure determination and inhibitor development efforts, which may lead to the evolution of promising and effective treatments for Alzheimer's disease.
