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Design, synthesis, and assembly of genetically engineered proteins: Simple routes to biocatalytic surfaces
Abstract
Hybrid artificial proteins, in which artificial repetitive polypeptides are fused with natural proteins or subunits, are proposed as a new approach to combining materials properties with the biological functions of natural proteins. As a model system, we have produced hybrid artificial proteins as "sticky enzymes" where the artificial domain serves to bind to surfaces while the natural domain performs catalytic activities. The artificial domains are repetitive polypeptides designed to provide multiple acidic functional groups and/or adopt regular secondary structures. A family of artificial proteins with sequence motifs-(-(AlaGly)$\sb{\rm x}$ZGly-) -$\sb{\rm n}$ (Z = Glu or ProGlu) is used to gain control over the materials properties of the hybrid. The natural domain is a bacterial phosphotriesterase which catalyzes rapid hydrolysis of organophosphorus pesticides and nerve agents. Hybrid artificial proteins are produced by fusion of genes encoding the artificial and enzyme domains through recombinant DNA technology. The target proteins, with molecular weights varied from 55 to 65 kDa, were expressed in Escherichia coli. All the hybrids, even in crude cell lysates, exhibit catalytic activity for hydrolysis of paraoxon (diethyl p-nitrophenyl phosphate). The hybrid that contains the artificial domain ((AlaGly)$\sb3$ProGluGly) $\sb{16}$ (designated HAP3PEG16) exhibits Michaelis-Menten kinetics, with K$\sb{\rm m}$ = 120 $\mu$M and k$\sb{\rm cat}$ = 2500 s$\sp{-1}$. The hybrid artificial proteins are selectively adsorbed on basic surfaces (e.g., DEAE Sephadex) through the anionic charges of the artificial domain, and elute only at NaCl $\geq$ 100 mM. Immobilized HAP3PEG16 exhibits k$\sb{\rm cat}$ of 1800 s$\sp{-1}$ when using Protein-Pak DEAE microspheres as the insoluble support, yielding 72% relative activity compared with the free soluble form judging from the k$\sb{\rm cat}$ value. In a related investigation, a recombinant phosphotriesterase with six consecutive histidine residues at the N-terminus was constructed and expressed in Escherichia coli under control of a T7 phage promoter. The hexahistidine sequence allows purification of the recombinant enzyme by metal chelate affinity chromatography. The recombinant enzyme catalyzes hydrolysis of paraoxon with a k$\sb{\rm cat}$ of 3880 s$\sp{-1}$ and K$\sb{\rm m}$ of 150 $\mu$M. The recombinant phosphotriesterase was immobilized on non-porous silica microspheres by physical adsorption at 34 units of enzyme activity per mg of support. The immobilized phosphotriesterase exhibits a catalytic rate constant of 3490 s$\sp{-1}$. A new method using a continuous-flow biosensor arrangement is proposed for direct spectrophotometric determination of organophosphate pesticides. The method has been demonstrated by immobilization of the recombinant phosphotriesterase on glass beads by physical adsorption and catalytic hydrolysis of paraoxon to product p-nitrophenolate, which can be quantitatively analyzed by electronic absorption spectroscopy. A low detection limit (5 ng/ml), wide linear range (25 to 2500 ng/ml), good precision (R.S.D.= 0.7-5.8%) and sample frequency of 24 samples per hour were achieved by the proposed method. (Abstract shortened by UMI.)
Subject Area
Polymers|Environmental science|Genetics|Biochemistry|Chemical engineering|Analytical chemistry
Recommended Citation
Wu, Dong, "Design, synthesis, and assembly of genetically engineered proteins: Simple routes to biocatalytic surfaces" (1997). Doctoral Dissertations Available from Proquest. AAI9809413.
https://scholarworks.umass.edu/dissertations/AAI9809413