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Results for “Protein Engineering”

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At least 379 records · Page 21Linked to original sources
PubMed1992

Protein engineering of Drosophila alcohol dehydrogenase. The hydroxyl group of Tyr152 is involved in the active site of the enzyme.

Drosophila alcohol dehydrogenase is the most studied member of the family of short-chain alcohol dehydrogenases, although its tridimensional structure still remains unknown. We have engineered a Drosophila alcohol dehydrogenase in which tyrosine-152, an invariant residue in all members of the family, has been substituted by phenylalanine. The mutated gene has been expressed in yeast and pure mutant enzyme has been prepared by a one-step FPLC chromatographic procedure. Drosophila alcohol dehydrogenase-phenylalanine-152 shows no enzymatic activity. This result suggests not only that tyrosine-152 could constitute an essential building block of the active site but also that its hydroxyl group is directly involved in the redox reaction catalyzed by the enzyme.

Alcohol Dehydrogenase↗
PubMed1994

Locating ligand-binding sites in 7TM receptors by protein engineering.

Over the past year, mutational analysis of peptide receptors has started to change our understanding of the interaction between G protein coupled receptors and their ligands, an area previously almost totally dominated by results from studies of monoamine receptors. A picture is currently emerging, in which small ligands appear to bind in three (more or less) overlapping ligand-binding pockets in between the transmembrane segments. In contrast, contact residues for peptide and protein ligands have mainly been found in exterior regions of peptide and protein receptors. It is also becoming increasingly clear that agonists and antagonists may interact in vastly different manners, even though they are competitive ligands for a common receptor.

Binding Sites↗
PubMed2004

Periplasmic binding proteins: a versatile superfamily for protein engineering.

The diversity of biological function, ligand binding, conformational changes and structural adaptability of the periplasmic binding protein superfamily have been exploited to engineer biosensors, allosteric control elements, biologically active receptors and enzymes using a combination of techniques, including computational design. Extensively redesigned periplasmic binding proteins have been re-introduced into bacteria to function in synthetic signal transduction pathways that respond to extracellular ligands and as biologically active enzymes.

Computer-Aided Design↗
PubMed1992

Protein engineering of the milk-clotting aspartic proteinases.

Calf Chymosin and a fungal protease from Mucor pusillus (Mucor rennin) are members of the aspartic proteinases used as milk-coagulants in cheese industry. A system for production of recombinant chymosin as inclusion bodies in Escherichia coli cells and its refolding into the active form was established. Another expression system for production of Mucor rennin in Saccharomyces cerevisiae was also established. Mucor rennin was efficiently excreted from the yeast host as a heavily glycosylated form. Glycosylation affected both the secretion and the enzyme properties. Site-directed mutagenesis of the Tyr residue at position 75 in chymosin and Mucor rennin revealed its crucial role in catalytic function of the aspartic proteinases. The results also suggested possibility to improve practical properties of the milk-clotting enzymes by site-directed mutagenesis.

Amino Acid Sequence↗
PubMedDate not supplied

[Protein engineering of uridine phosphorylase from Escherichia coli K-12. II. Comparative study of hybrid and mutant forms of uridine phosphorylases].

Genes for hybrid uridine phosphorylases (UPases) consisting of fragments of amino acid sequences of UPases from Escherichia coli and Salmonella typhimurium were constructed. Producing strains of the corresponding proteins were genetically engineered. Mutant forms of the E. coli K-12 UPase were produced by site-directed mutagenesis. A comparative study of the enzyme properties of the mutant and hybrid forms of bacterial UPases was performed. It was shown that Asp27 unlike Asp5 and Asp29 residues of the E. coli UPase forms part of the active site of the protein. A scheme of the involvement of Asp27 in the binding of inorganic phosphate is proposed.

Amino Acid Sequence↗
PubMed1998

Investigation of functional aspects of the N-terminal region of elongation factor Tu from Escherichia coli using a protein engineering approach.

The function of the N-terminal region of elongation factor Tu is still unexplained. Until recently, it has not been visible in electron density maps from x-ray crystallography studies, but the presence of several well conserved basic residues suggest that this part of the molecule is of structural importance for the factor to function properly. In this study, two lysines at positions 4 and 9 were mutated separately to alanine or glutamate. The resulting four point mutants were expressed and purified using the pGEX system. The untagged products were characterized with regard to guanine-nucleotide interaction, intrinsic GTPase activity, and binding of aminoacyl-tRNA (aa-tRNA). The results show that Lys9 is especially strongly involved in the association with guanine nucleotides and the binding of aa-tRNA. Also Lys4 plays a role in the association of GDP and GTP and is also of some importance in aa-tRNA binding. Our results are discussed in structural terms with the conclusion that a complex network of interactions across the interface between domains 1 and 2 with Lys9 being a key residue seems to be important for the fine tuning of the dimensions of the cleft accommodating the acceptor end of aa-tRNA as well as delineating the structure of the effector region.

Escherichia coli↗
PubMed1996

Transgenic barley expressing a protein-engineered, thermostable (1,3-1,4)-beta-glucanase during germination.

The codon usage of a hybrid bacterial gene encoding a thermostable (1,3-1,4)-beta-glucanase was modified to match that of the barley (1,3-1,4)-beta-glucanase isoenzyme EII gene. Both the modified and unmodified bacterial genes were fused to a DNA segment encoding the barley high-pI alpha-amylase signal peptide downstream of the barley (1,3-1,4)-beta-glucanase isoenzyme EII gene promoter. When introduced into barley aleurone protoplasts, the bacterial gene with adapted codon usage directed synthesis of heat stable (1,3-1,4)-beta-glucanase, whereas activity of the heterologous enzyme was not detectable when protoplasts were transfected with the unmodified gene. In a different expression plasmid, the codon modified bacterial gene was cloned downstream of the barley high-pI alpha-amylase gene promoter and signal peptide coding region. This expression cassette was introduced into immature barley embryos together with plasmids carrying the bar and the uidA genes. Green, fertile plants were regenerated and approximately 75% of grains harvested from primary transformants synthesized thermostable (1,3-1,4)-beta-glucanase during germination. All three trans genes were detected in 17 progenies from a homozygous T1 plant.

Amino Acid Sequence↗
PubMed1991

Protein engineering to change thermal stability for food enzymes.

In this review we have briefly indicated how the present state of knowledge allows proteins to be mutated to increase or decrease stability. We have discussed experiments on both model proteins and those of relevance to the food industry, and show how hydrophobic forces are a major driving force for folding as well as having a major role in thermostability. We have also indicated the large contribution that hydrogen bonding, electrostatic interactions and, in a less well predicted way, disulphide bridges make to thermostability.

Enzyme Stability↗
PubMed2006

Protein engineering of class-A non-specific acid phosphatase (PhoN) of Salmonella typhimurium: modulation of the pH-activity profile.

Engineering of the PhoN enzyme of Salmonella typhimurium due to its superior characteristics for bioremediation of heavy metals has been advocated by Macaskie and colleagues [Basnakova, G., Stephens, E.R., Thaller, M.C., Rossolini, G.M., Macaskie, L.E., 1998. The use of Escherichia coli bearing a phoN gene for the removal of uranium and nickel from aqueous flows. Appl. Microbiol. Biotechnol. 50, 266-272]. The native enzyme hydrolyzes disparate organophosphates and exhibits optimal phosphatase activity at pH 5.5, for instance, with substrate p-nitrophenyl phosphate. Structurally guided Ile-78 was mutated using site-directed mutagenesis to Ala, Asp and His residues, with an aim to shift the optimum pH of the PhoN enzyme. Encouragingly, the I78A mutant displays significantly higher (as high as 160%) enzymatic efficiency over a broad pH range of 3.0-9.0, compared to the wild-type PhoN. The higher catalytic efficiency is due to the increase in k(cat), and can be mainly attributed to a deshielding of catalytic His-158 from the bulk-solvent. The I78D mutant possesses nearly twice the specific activity at the optimum pH of 7.0. The alkaline shift of the pH-activity profile agrees well with reasoning based on electrostatics. An increase in K(m), however, lowers the catalytic efficiency of the I78D mutant at the optimum pH. The I78H mutant, counter-intuitively, also exhibits an alkaline shift in the pH-optimum. Nonetheless, the active site scaffold in I78H mutant may not be disturbed, as similar steady-state kinetic parameters are observed for both I78H mutant and wild-type PhoN at their respective pH optima.

Acid Phosphatase↗
PubMed2005

Four-base codon/anticodon strategy and non-enzymatic aminoacylation for protein engineering with non-natural amino acids.

Techniques for position-specific incorporation of non-natural amino acids in an in vitro protein synthesizing system are described. First, a PNA-assisted non-enzymatic tRNA aminoacylation with a variety of natural and non-natural amino acids is described. With this technique, one can aminoacylate a specific tRNA simply by adding a preformed amino acid activated ester-PNA conjugate into an in vitro protein biosynthesizing system. Second, the genetic code is expanded by introducing 4-base codons that can be exclusively translated to non-natural amino acids. The most advantageous point of the 4-base codon strategy is to introduce multiple amino acids into specific positions in single proteins by using mutually orthogonal 4-base codons and orthogonal tRNAs. An easy and quick method for preparation of tRNAs possessing 4-base anticodons is also described. Combination of the non-enzymatic aminoacylation and the 4-base codon/anticodon strategy gives an easy and widely applicable technique for incorporating a variety of non-natural amino acids into proteins in vitro.

Amino Acids↗
PubMed1995

[Study of the structure and mechanism of action of troponin C and calmodulin by "protein" engineering].

The structure, properties and action mechanisms of troponin C and calmodulin are reviewed. The primary and tertiary structures of calcium-binding proteins and the mechanisms of Ca2+ binding are analyzed. The methods used for investigating the functional activity of Ca-binding proteins are compared. Molecular biology approaches for analyzing the role of various ligands in Ca2+ binding are described. The role of alpha-helices in the maintenance of the overall structure, Ca2+ binding and calmodulin and troponin C interaction with target proteins is discussed. Mutations directed at the change of the electric charge and hydrophobicity of calmodulin and troponin C are described and compared. Data on the incorporation of Cys residues into calmodulin and troponin C structure are presented. The use of Cys-containing mutants of Ca-binding proteins for the study of conformational changes and protein-protein interaction is analyzed.

Amino Acid Sequence↗