5/7/2023 0 Comments Puc19 snapgene![]() ![]() Four cysteine residues create disulfide bridges linking the two subunits and are essential for mature BAP dimer activity. In the fully active, dimeric BAP, Zn 2+ occupies active sites A and B, and Mg 2+ occupies site C, thus the enzyme has the configuration (ZnAZnBMgC)2. Furthermore, they require the adoption of a catalytically active conformation facilitated by disulfide bridges, among others. Their common architecture includes each catalytic site containing three metal ions: two Zn 2+ and one Mg 2+. APS are almost exclusively homodimeric metalloproteins. ![]() Such a dedicated system was described as the phosphate-specific transport system (Pst system). coli utilises a dedicated permease for the transport of this ion through its internal membrane - a nonpolar region essentially impermeable to charged molecules. Since phosphate is a highly charged anion, E. coli bacteria contain a double membrane, where the outer membrane is decorated with porin proteins, thus allowing for the diffusion of charged molecules. Once a phosphate group is clipped from a variety of organic compounds, it needs to enter the cytoplasm. coli BAP is to supply a source of inorganic phosphate when the environment is deprived of this compound by increasing the rate of diffusion of this compound into the cells and preventing phosphate from leaving the cells. Further, the method of transient inactivation of secreted toxic enzymes by conducting their biosynthesis in an inactive state in the cytoplasm, followed by in vitro reactivation, can be generally applied to other problematic proteins.Īlkaline phosphatases (APS) (EC 3.1.3.1) are enzymes commonly found in nature, from bacteria to mammals. The developed method appears well suited for the industrial production of ultrapure BAP. The reconstituted His6-BAP exhibited high activity and was used to develop an efficient protocol for all types of DNA termini, including problematic ones (blunt, 3′-protruding). A simple protocol based on precipitation and immobilized metal affinity chromatography (IMAC) purification yielded homogeneous protein, which was reactivated by dialysis into a redox buffer containing reduced and oxidized sulfhydryl group compounds, as well as the protein structure stabilizing cofactors Zn 2+, Mg 2+ and phosphate. coli cytoplasm, preventing formation of the active enzyme. The His6-BAP protein was harmless to the cells, as its natural toxicity was inhibited by the reducing environment within the E. The gene expression was robust, resulting in accumulation of His6-BAP in the cytoplasm, exceeding 50% of total cellular proteins. coli in a P BAD promoter expression vector. The recombinant bap gene was modified by replacing a secretion leader coding section with a N-terminal His6-tag, cloned and expressed in E. We present a novel approach to phoA gene cloning, engineering, expression, purification and reactivation of the transiently inactivated enzyme. Then, the leader is clipped off and dimers are formed upon oxidation. The precursor enzyme contains a signal peptide at the N-terminus and is secreted to the E. As it removes terminal phosphate from DNA, RNA and deoxyribonucleoside triphosphates, it is used to lower self-ligated vectors’ background. coli) is a key enzyme used in protein/antibody detection and molecular cloning. Bacterial alkaline phosphatase (BAP) from Escherichia coli ( E. An industrial approach to protein production demands maximum possible yields of biosynthesized proteins, balanced with the recombinant host’s viability. Various approaches have been taken to overcome these limitations, exemplified by tight control of recombinant genes or secretion of recombinant proteins. The biotechnology production of enzymes is often troubled by the toxicity of the recombinant products of cloned and expressed genes, which interferes with the recombinant hosts’ metabolism. ![]()
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