Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Protein Engineering”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 217 records · Page 12Linked to original sources
PubMed2004

Lattice stabilization and enhanced diffraction in human p38 alpha crystals by protein engineering.

Mitogen-activated protein (MAP) kinase p38 alpha is activated in response to environmental stress and cytokines, and plays a significant role in inflammatory responses. For these reasons, it is an important target for the treatment of a wide range of inflammatory and autoimmune diseases. The crystals of p38 alpha that we obtained by published procedures were usually small, quite mosaic, and difficult to reproduce and thus posed a difficulty for the intensive high-resolution studies required for a structure-guided drug discovery approach. Based on crystallographic and biochemical evidences, we prepared a single point mutation of a surface cysteine (C162S) and found that it prevents aggregation and improves the homogeneity and stability of the enzyme. This mutation also facilitates the crystallization process and increases the diffracting power of p38 alpha crystals. Surprisingly, we found that the mutation induces a change in the conformation of a nearby surface loop resulting in stronger lattice interactions, consistent with the improved crystal quality. The mutant protein, because of its improved stability and strengthened lattice interactions, thus provides a significantly improved reagent for use in structure-based drug design for this important disease target.

Crystallization↗
PubMed2006

Oxidative stabilization of iso-1-cytochrome c by redox-inspired protein engineering.

Iso-1-cytochrome c, as any other hemeprotein, is able to react with hydrogen peroxide and to engage in the peroxidase cycle. However, peroxidases are irreversibly inactivated by their substrate, hydrogen peroxide. The oxidative inactivation of hemeproteins is mechanism based and arises as the consequence of unproductive electron abstraction reactions. Protein elements, such as the porphyrin ring or the protein backbone, act as simultaneous and competing electron sources even in the presence of exogenous reducing substrates, leading to a decline in activity. It is hypothetically possible to alter the intramolecular electron transfer pathways by direct replacement of low redox potential residues around the active site; as a consequence, the inactivation process would be delayed or even suppressed. To demonstrate this hypothesis, a redox-inspired strategy was implemented until an iso-1-cytochrome c variant fully stable at catalytic concentrations of hydrogen peroxide was obtained. This variant, harboring the N52I,W59F,Y67F,K79A,F82G substitutions, preserved the catalytic performance of the parental protein but achieved a 15-fold higher total-turnover number. The phenotype of this variant was reflected in the stability of its electronic components, allowing identification of a protein-based radical intermediate mechanistically similar to Compound I of classical peroxidases. The results presented here clearly demonstrate that redox-inspired protein engineering is a useful tool for the rational modulation of intramolecular electron transfer networks.

Amino Acid Sequence↗
PubMed2004

Protein engineering of cytochrome b562 for quinone binding and light-induced electron transfer.

The central photochemical reaction in photosystem II of green algae and plants and the reaction center of some photosynthetic bacteria involves a one-electron transfer from a light-activated chlorin complex to a bound quinone molecule. Through protein engineering, we have been able to modify a protein to mimic this reaction. A unique quinone-binding site was engineered into the Escherichia coli cytochrome b(562) by introducing a cysteine within the hydrophobic interior of the protein. Various quinones, such as p-benzoquinone and 2,3-dimethoxy-5-methyl-1,4-benzoquinone, were then covalently attached to the protein through a cysteine sulfur addition reaction to the quinone ring. The cysteine placement was designed to bind the quinone approximately 10 A from the edge of the bound porphyrin. Fluorescence measurements confirmed that the bound hydroquinone is incorporated toward the protein's hydrophobic interior and is partially solvent-shielded. The bound quinones remain redox-active and can be oxidized and rereduced in a two-electron process at neutral pH. The semiquinone can be generated at high pH by a one-electron reduction, and the midpoint potential of this can be adjusted by approximately 500 mV by binding different quinones to the protein. The heme-binding site of the modified cytochrome was then reconstituted with the chlorophyll analogue zinc chlorin e(6). By using EPR and fast optical techniques, we show that, in the various chlorin-protein-quinone complexes, light-induced electron transfer can occur from the chlorin to the bound oxidized quinone but not the hydroquinone, with electron transfer rates in the order of 10(8) s(-1).

Binding Sites↗
PubMed1991

Folding around the C-terminus of human carbonic anhydrase II. Kinetic characterization by use of a chemically reactive SH-group introduced by protein engineering.

We are characterizing the process of refolding of the enzyme human carbonic anhydrase II from the denatured state in guanidine hydrochloride. To describe the folding in defined parts of the protein we use protein engineering to introduce cysteine residues as unique chemically reactive probes. The accessibility of the cysteine SH-group to the alkylating reagent iodoacetate, at different stages during refolding, is used to give a kinetic description of the folding process. The structuration of the C-terminal part of the polypeptide chain, which is involved in a unique 'knot' topology, was investigated. Our results show that the structure around the C-terminal, composed of the outermost beta-strands in a dominating beta-structure that extends through the entire protein, is formed relatively late during refolding. In contrast, it was found that beta-strands located in the interior of the protein were structured very rapidly. The final native structure is formed in a process that is slower than those observed for formation of beta-structure.

Carbonic Anhydrases↗
PubMed1991

Conversion of pig pancreas phospholipase A2 by protein engineering into enzyme active against Escherichia coli treated with the bactericidal/permeability-increasing protein.

Phospholipases A2 (PLA-2) are conserved enzymes that can vary widely in their activity toward certain biological targets. Activity of PLA-2 toward Escherichia coli treated with the bactericidal/permeability-increasing protein (BPI) of granulocytes has been detected only in "Group II" PLA-2 (lacking Cys11-Cys77) and correlates with overall basicity and the presence of a cluster of basic amino acids within a variable surface region near the NH2 terminus (including residues 6, 7, 10, 11, and 15). We now show that of five pancreatic PLA-2 ("Group I" enzymes) tested from different species of mammals, the human enzyme that is most basic both globally (pI 8.7) and locally (Arg-6, Lys-7, and Lys-10) is active toward BPI-treated E. coli (approximately 1-2% activity of the most active Group II PLA-2) whereas the other four PLA-2 are essentially inactive (less than 0.1%). The cDNA of the pig pancreatic PLA-2 (pI 6.4; Arg-6, Ser-7, Lys-10) has been modified by site-specific mutagenesis and the wild-type and mutant PLA-2 have been expressed in and purified from either E. coli or Saccharomyces cerevisiae to determine more precisely the structural determinants of PLA-2 activity toward BPI-treated E. coli. The single substitution of lysine (or arginine) for Ser-7 transformed the pig pancreatic PLA-2 into an active enzyme toward BPI-treated E. coli possessing 25-50% the activity of the human PLA-2. Additional modifications to increase global basicity (increase in net charge up to +4) caused a further (up to 2-fold) increase in activity. All mutant PLA-2 still containing Ser-7 possessed little or no activity toward BPI-treated E. coli. Changes in activity toward BPI-treated E. coli were accompanied by parallel changes in enzyme binding to this target. In contrast, substitution of lysine (or arginine) for Ser-7 caused little or no alteration of enzyme activity toward either autoclaved E. coli or egg yolk lipoproteins indicating no major effects on the catalytic properties of the PLA-2. This study demonstrates directly the role of NH2-terminal basic residues in the action of PLA-2 on BPI-treated E. coli and suggests that these properties mainly facilitate PLA-2 binding to this biological target.

Animals↗
PubMed2005

An on-pathway hidden intermediate and the early rate-limiting transition state of Rd-apocytochrome b562 characterized by protein engineering.

The folding pathway of Rd-apocytochrome b562, a four-helix bundle protein, was characterized using Trp and Ala/Gly pair mutations. We found that the Trp mutants (F65W) of both the fully folded Rd-apocytochrome b562 and a partially unfolded intermediate with the N-terminal helix (helix I) unfolded, fold with identical folding rates, providing direct evidence for the conclusion that the rate-limiting transition state folds before the partially unfolded intermediate; and that this hidden intermediate is an on-pathway intermediate. We further characterized the helical structures formed in the rate-limiting transition state by measuring the folding/unfolding rates for Ala/Gly pair mutations at solvent-exposed positions. Little change in folding rates occurred for the Ala/Gly pair mutations at positions in helix I and the C-terminal regions of helix II and IV. In contrast, a significant difference in folding rates was observed for the Ala/Gly pair mutations in helix III and the N-terminal regions of helix II and IV, suggesting that helix III and the N-terminal regions of helix II and IV are formed in the rate-limiting transition state. These results complement those obtained from earlier studies and help to define the folding pathway of Rd-apocytochrome b562 in more detail.

Alanine↗
PubMed2002

Protein engineering and properties of human metalloproteinase and thrombospondin 1.

This work generated many truncated proteins and Glu(385) to Ala (E(385)/A) mutants of the human metalloproteinase and thrombospondin 1 (METH-1 or ADAMTS1) and specific antibodies. METH-1 was an active endopeptidase and both the metalloproteinase and the disintegrin/cysteine-rich domains were required for the proteinase activity. A point mutation at the zinc-binding site (E(385)/A) abolished the catalytic activity. METH-1 protein function may be modulated through proteolytic cleavage at multiple sites. One 135 kDa species had an NH(2)-terminal sequence of L(33)GRPSEEDEE. A species at 115 kDa and some other protein bands began with F(236)VSSHRYV(243), indicating that METH-1 proenzyme might be activated by a proprotein convertase such as furin by cleaving the R(235)-F(236) peptide bond. This cleavage was not an autocatalytic process since the E(385)/A mutants were also processed. Furthermore, a 52 kDa band with an NH(2)-terminal sequence of L(800)KEPLTIQV resulted from the digestion between the first and the second thrombospondin 1-like motifs in the spacer region of the extracellular matrix-binding domains.

ADAM Proteins↗
PubMed1998

Protein engineering and the development of generic biosensors.

Biosensors exploit the remarkable specificity of biomolecular recognition to provide analytical tools that can measure the presence of a single molecular species in a complex mixture. A new strategy is emerging in the development of biosensor technologies: molecular-engineering techniques are being used to adapt the properties of proteins to simple, generic detector instrumentation, rather than adapting instruments to the unique requirements of a natural molecule.

Binding Sites↗
PubMedDate not supplied

Improved yield and stability of L49-sFv-beta-lactamase, a single-chain antibody fusion protein for anticancer prodrug activation, by protein engineering.

The L49 single-chain Fv fused to beta-lactamase (L49-sFv-bL) combined with the prodrug C-Mel is an effective anticancer agent against tumor cells expressing the p97 antigen. However, large-scale production of L49-sFv-bL from refolded E. coli inclusion bodies has been problematic due to inefficient refolding and instability of the fusion protein. Sequence analysis of the L49-sFv framework regions revealed three residues in the framework regions at positions L2, H82B, and H91, which are not conserved for their position, occurring in <1% of sequences in Fv sequence databases. One further unusual residue, found in <3% of variable sequences, was observed at position H39. Each unusual residue was mutated to a conserved residue for its position and tested for refolding yield from inclusion bodies following expression in E. coli. The three V(H) single mutants showed improvement in the yield of active protein and were combined to form double and triple mutants resulting in a 7-8-fold increased yield compared to the parental protein. In an attempt to further improve yield, the orientation of the triple mutant was reversed to create a bL-L49-sFv fusion protein resulting in a 3-fold increase in expressed inclusion body protein and producing a 20-fold increase in the yield of purified protein compared to the parental protein. The triple mutants in both orientations displayed increased stability in murine plasma and binding affinity was not affected by the introduced mutations. Both triple mutants also displayed potent in vitro cytotoxicity and in vivo antitumor activity against p97 expressing melanoma cells and tumor xenografts, respectively. These results show that a rational protein-engineering approach improved the yield, stability, and refolding characteristics of L49-sFv-bL while maintaining binding affinity and therapeutic efficacy.

Animals↗
PubMed1997

Protein engineering from a bioindustrial point of view.

Work with proteins, particularly enzymes, is a rapidly growing segment of the biotechnology industry. Directed evolution promises to become an increasingly important strategy in their development as it allows one to sidestep some of the difficult questions relating the structural and functional properties of such proteins to their industrial utility. It is also clear, however, that greater understanding of how to engineer certain basic enzyme properties, such as stability, activity, and surface properties, is beginning to emerge, and this understanding will make rational design more efficient. To engineer a commercially useful protein many properties need to be changed, and frequently these changes are interdependent. Recent protein engineering studies on protease, amylase, lipase and cellulase illustrate some of the progress in this area.

Journal Article↗
PubMed1992

Protein engineering for molecular electronics.

Recombinant DNA technology allows the manipulation of the physical properties of proteins that perform electron transport and photochemical processes. Recent work is reviewed that has a potential impact on the development of molecular electronic devices, within a general framework outlining strategies for device fabrication. This review is also published in Current Opinion in Structural Biology 1992, 2:587-592.

Bacteriorhodopsins↗
PubMed1990

Improvement of nutritional value and functional properties of soybean glycinin by protein engineering.

Glycinin is one of the predominant storage proteins of soybean. To improve its functional properties (heat-induced gelation and emulsification) and/or nutritional value, the A1aB1b proglycinin subunit was modified on the basis of genetically variable domains suggested from the comparison of amino acid sequences of glycinin-type globulins from various legumes and nonlegumes and the relationships between the structure and the functional properties of glycinin. Thus, nucleotide sequences corresponding to each of the variable domains were deleted from the cDNA encoding the A1aB1b proglycinin, and a synthetic DNA encoding four continuous methionines was inserted into the cDNA region corresponding to each of the variable domains. Expression plasmids carrying the modified cDNAs were constructed and expressed in Escherichia coli strain JM105. Some of the modified proteins were accumulated as soluble proteins in the cells at a high level and self-assembled. They exhibited functional properties superior to those of the native glycinin from soybean, which establishes the possibility of creating theoretically designed novel glycinins with high food qualities.

Amino Acid Sequence↗
PubMed1989

Plasma desorption mass spectrometry, an analytical tool in protein engineering: characterization of modified insulins.

Californium-252 plasma desorption mass spectrometry (PDMS) has been employed for the characterization of a series of human insulin derivatives in order to evaluate the performance of this technique as an analytical tool in protein engineering. Several of the characterized modifications result in a 1 a.m.u. mass change. The precision in mass determination obtainable by PDMS analysis is not sufficient for unambiguous verification of such modifications based on the molecular weight alone. It is, however, possible to carry out in situ enzymatic digestion of the sample. Subsequent PDMS analysis will in most cases reveal if the modification has been introduced as intended.

Amino Acid Sequence↗