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At least 721 records · Page 40Linked to original sources
PubMed2001

Development and application of a biological peptide pump for the study of the in vivo actions of angiotensin peptides.

In the past few years, a great deal of interest has been focused on the possibility that angiotensin peptides could have direct effects on target tissues independent of their hemodynamic effects. In addition, there has been much speculation on the potential biological roles of angiotensin peptides other than angiotensin II. Unfortunately, a direct test of these possibilities in whole animals has been difficult due to limitations in existing biological systems. In this review, we describe the characteristics of an engineered protein capable of directing the production of a wide variety of peptides to specific tissues and cell types in transgenic animals and discuss its potential applications.

Angiotensins↗
PubMed2004

Enhancement of correct protein folding in vivo by a non-lytic baculovirus.

The BEVS (baculovirus expression vector system) is widely used for the production of proteins. However, engineered proteins frequently experience the problem of degradation, possibly due to the lytic nature of the conventional BEVS (herein referred to as L-BEVS). In the present study, a non-lytic BEVS (N-BEVS) was established by random mutagenesis of viral genomes. At 5 days post-infection, N-BEVS showed only 7% cell lysis, whereas L-BEVS showed 60% lysis of cells. The quality of protein expressed in both N- and L-BEVSs was examined further using a novel FRET (fluorescence resonance energy transfer)-based assay. To achieve this, we constructed a concatenated fusion protein comprising LUC (luciferase) sandwiched between EYFP (enhanced yellow fluorescent protein) and ECFP (enhanced cyan fluorescent protein). The distance separating the two fluorescent proteins in the fusion protein EYFP-LUC-ECFP (designated hereafter as the YLC construct) governs energy transfer between EYFP and ECFP. FRET efficiency thus reflects the compactness of LUC, indicating its folding status. We found more efficient FRET in N-BEVS compared with that obtained in L-BEVS, suggesting that more tightly folded LUC was produced in N-BEVS. YLC expression was also analysed by Western blotting, revealing significantly less protein degradation in N-BEVS than in L-BEVS, in which extensive degradation was observed. This FRET-based in vivo folding technology showed that YLC produced in N-BEVS is more compact, correlating with improved resistance to degradation. N-BEVS is thus a convenient alternative for L-BEVS for the production of proteins vulnerable to degradation using baculoviruses.

Animals↗
PubMed1994

Optimal sequence selection in proteins of known structure by simulated evolution.

Rational design of protein structure requires the identification of optimal sequences to carry out a particular function within a given backbone structure. A general solution to this problem requires that a potential function describing the energy of the system as a function of its atomic coordinates be minimized simultaneously over all available sequences and their three-dimensional atomic configurations. Here we present a method that explicitly minimizes a semiempirical potential function simultaneously in these two spaces, using a simulated annealing approach. The method takes the fixed three-dimensional coordinates of a protein backbone and stochastically generates possible sequences through the introduction of random mutations. The corresponding three-dimensional coordinates are constructed for each sequence by "redecorating" the backbone coordinates of the original structure with the corresponding side chains. These are then allowed to vary in their structure by random rotations around free torsional angles to generate a stochastic walk in configurational space. We have named this method protein simulated evolution, because, in loose analogy with natural selection, it randomly selects for allowed solutions in the sequence of a protein subject to the "selective pressure" of a potential function. Energies predicted by this method for sequences of a small group of residues in the hydrophobic core of the phage lambda cI repressor correlate well with experimentally determined biological activities. This "genetic selection by computer" approach has potential applications in protein engineering, rational protein design, and structure-based drug discovery.

Algorithms↗
PubMed2000

ASAP: analysis of peptide composition.

SUMMARY: ASAP is a web tool designed to search for specific dipeptides, tripeptides and tetrapeptides in a protein sequence database. The server allows for: (a) identification of frequent and infrequent peptides and the creation of peptide probability tables for a given database of sequences (GenerNet program), (b) determination of the compatibility of an amino-acid sequence to the given peptide probability tables (ClonErrNet program); and (c) comparison of different protein databases based on peptide composition (CompNet program). ASAP server can be useful in protein engineering and/or protein classification studies.

Computational Biology↗
PubMed2006

Improved Pruning algorithms and Divide-and-Conquer strategies for Dead-End Elimination, with application to protein design.

MOTIVATION: Structure-based protein redesign can help engineer proteins with desired novel function. Improving computational efficiency while still maintaining the accuracy of the design predictions has been a major goal for protein design algorithms. The combinatorial nature of protein design results both from allowing residue mutations and from the incorporation of protein side-chain flexibility. Under the assumption that a single conformation can model protein folding and binding, the goal of many algorithms is the identification of the Global Minimum Energy Conformation (GMEC). A dominant theorem for the identification of the GMEC is Dead-End Elimination (DEE). DEE-based algorithms have proven capable of eliminating the majority of candidate conformations, while guaranteeing that only rotamers not belonging to the GMEC are pruned. However, when the protein design process incorporates rotameric energy minimization, DEE is no longer provably-accurate. Hence, with energy minimization, the minimized-DEE (MinDEE) criterion must be used instead. RESULTS: In this paper, we present provably-accurate improvements to both the DEE and MinDEE criteria. We show that our novel enhancements result in a speedup of up to a factor of more than 1000 when applied in redesign for three different proteins: Gramicidin Synthetase A, plastocyanin, and protein G. AVAILABILITY: Contact authors for source code.

Algorithms↗
PubMed2003

Beta-propellers: associated functions and their role in human diseases.

The beta-propeller fold appears as a very fascinating architecture based on four-stranded antiparallel and twisted beta-sheets, radially arranged around a central tunnel. Similar to the alpha/beta-barrel (TIM-barrel) fold, the beta-propeller has a wide range of different functions, and is gaining substantial attention. Some proteins containing beta-propeller domains have been implicated in the pathogenesis of a variety of diseases such as cancer, Alzheimer, Huntington, arthritis, familial hypercholesterolemia, retinitis pigmentosa, osteogenesis, hypertension, and microbial and viral infections. This article reviews some aspects of 3D structure, amino acids sequence regularities, and biological functions of the proteins containing beta-propeller domains. Major emphasis has been laid on beta-propellers whose functions are associated to human diseases. Recent research efforts reported in the fields of protein engineering, drug design, and protein structure-function relationship studies, concerning the beta-propeller architecture, have also been discussed.

Disease↗
PubMed1997

Enhanced anti-tumor effect of an IFN-gamma-EGF fusion protein.

The novel fusion proteins harboring human or mouse interferon combined with epidermal growth factor receptor binding domain were constructed using methods of genetic and protein engineering. The fusion proteins were assayed to retain complete antiviral activities. The EGF receptor binding moiety of the fusion proteins exhibited competitive binding against 125I-EGF for EGF receptors on A431 cells. The fusion proteins were shown to be more potent in inhibiting the growth of cultured target carcinoma cells than interferon-gamma alone. Experimental data derived from mouse B16 malignant melanoma models indicates that the weight of tumor in mice treated with IFN fusion proteins was significantly smaller than that of mice treated with interferon-gamma alone. The work here is unprecedented in the world and provides a reliable evidence to support the upcoming clinical employment of a class of interferons that specifically target tumor cell.

Amino Acid Sequence↗
PubMedDate not supplied

Engineering and characterization of a novel fusion protein incorporating B7.2 and an anti-ErbB-2 single-chain antibody fragment for the activation of Jurkat T cells.

The provision of the T-cell costimulatory molecule B7 to tumor cells can be an effective way to trigger a tumor-specific cytolytic T-cell response. One way to provide B7 to tumor cells would be to couple an antitumor antibody directly to B7. Such a molecule should target tumors displaying antigen and provide the costimulatory signal to T cells, resulting in the initiation of an antitumor T-cell response. To this end, a fusion protein was designed that incorporates a single-chain antibody fragment (scFv) to erbB-2 (Her2/neu), an oncogene product overexpressed by 30% to 50% of breast carcinomas, and the ECD of B7-2 (CD86). This fusion protein, expressed and purified from Pichia pastoris, was shown to retain binding activity to both counter receptors, erbB-2 and CD28. The fusion protein was also shown to target erbB-2-positive tumor cells and to deliver a CD28-specific T-cell costimulatory signal. These results suggest that a fusion protein engineered to target tumor cells and signal T cells for activation may be an effective means of cancer immunotherapy. Further studies should be performed to characterize the fusion protein in erbB-2 tumor-bearing mice for in vivo tumor targeting, biodistribution, and efficacy.

Amino Acid Sequence↗
PubMed1991

Review and patents and literature. The use of insect cell cultures for recombinant protein synthesis: Engineering aspects.

The use of the insect cell/baculovirus expression system for producing recombinant proteins of bacterial, plant, insect, and mammalian origin has become widespread. The popularity of this eukaryotic expression system is due to many factors, including (1) potentially high protein expression levels, (2) ease and speed of genetic engineering, (3) ability to accommodate large DNA inserts, (4) protein processing similar to higher eukaryotic cells (e.g., mammalia cells), and (5) ease of insect cell growth (e.g., suspension growth). The following review of the literature discusses two engineering aspects of recombinant protein synthesis by insect cell cultures: bioreactor scale-up and insect cell line selection. Following this review patent abstracts and additional literature pertaining to expression of recombinant proteins in insect cell culture are listed.

Animals↗
PubMed2002

Conjugation of luminescent quantum dots with antibodies using an engineered adaptor protein to provide new reagents for fluoroimmunoassays.

We describe the preparation and characterization of bioinorganic conjugates made with highly luminescent semiconductor CdSe-ZnS core-shell quantum dots (QDs) and antibodies for use in fluoroimmunoassays. The conjugation strategy employs an engineered molecular adaptor protein, attached to the QDs via electrostatic/hydrophobic self-assembly, to link the inorganic fluorophore with antibodies. In this method, the number of antibodies conjugated to a single QD can be varied. In addition, we have developed a simple purification strategy based on mixed-composition conjugates of the molecular adaptor and a second two-domain protein that allows the use of affinity chromatography. QD-antibody conjugates were successfully used in fluoroimmunoassays for detection of both a protein toxin (staphylococcal enterotoxin B) and a small molecule (2,4,6-trinitrotoluene).

Amino Acid Sequence↗
PubMed1988

Protein design for non-aqueous solvents.

Improving protein stability in unnatural and suboptimal environments is a promising application of protein engineering technology. Carefully designed amino acid alterations may lead to dramatic positive effects on the stability of proteins under highly perturbing conditions, such as in non-aqueous solvents. Applications of biocatalysts and proteins with specific binding capabilities in the chemical industry have been severely limited by constraints placed on the solvent environment. With the advent of convenient methods for altering the amino acid composition and even synthesizing entirely new protein molecules, it is worthwhile to consider engineering proteins for stability in non-aqueous solvents. In order to identify the features that a protein would need for stability in organic media, we have been studying the structure and properties of the hydrophobic protein crambin. Crambin is unique in that it is soluble and stable in very high concentrations of polar organic solvents. Crambin and its water-soluble homologs offer a powerful demonstration of protein engineering for non-aqueous solvents. This paper describes the structural features that contribute to crambin's special properties. Based on these observations and consideration of how non-aqueous solvents affect the interactions important in protein folding, a set of rules for designing non-aqueous solvent-stable proteins is proposed.

Amino Acid Sequence↗
PubMed2007

Design of a functional membrane protein by engineering a heme-binding site in glycophorin A.

We have designed a functional model membrane protein by engineering a bis-Histidine heme-binding site into a natural membrane protein, glycophorin A (GpA), structurally characterized by the dimerization of a single transmembrane helix. Out of the 32 residues comprising the transmembrane helix of GpA, five amino acids were mutated; the resulting protein, ME1, has been characterized in dodecyl phosphocholin (DPC) micelles by UV-vis, CD spectroscopy, gel electrophoresis, and analytical ultracentrifugation. ME1 binds heme with sub-micromolar affinity and maintains the highly helical secondary structure and dimeric oligomerization state of GpA. The ME1-Heme complex exhibits a redox potential of -128 +/- 2 mV vs SHE, indicating that the heme resides in a hydrophobic environment and is well shielded from the aqueous phase. Moreover, ME1 catalyzes the hydrogen peroxide dependent oxidation of organic substrates such as TMB (2,2',5,5'-tetramethyl-benzidine). This protein may provide a useful framework to investigate how the protein matrix tunes the cofactor properties in membrane proteins.

Amino Acid Sequence↗
PubMed2000

Anticalins versus antibodies: made-to-order binding proteins for small molecules.

Engineering proteins to bind small molecules presents a challenge as daunting as drug discovery, for both hinge upon our understanding of receptor-ligand molecular recognition. However, powerful techniques from combinatorial molecular biology can be used to rapidly select artificial receptors. While traditionally researchers have relied upon antibody technologies as a source of new binding proteins, the lipocalin scaffold has recently emerged as an adaptable receptor for small molecule binding. 'Anticalins', engineered lipocalin variants, offer some advantages over traditional antibody technology and illuminate features of molecular recognition between receptors and small molecule ligands.

Amino Acid Sequence↗
PubMed1992

Engineered metalloregulation in enzymes.

Protein engineering of metal-dependent enzyme activity is now possible due to the wealth of information available about metalloproteins. The results emerging from these studies provide insight into our understanding of the chemistry of metals in macromolecular environments as well as the biology of metal-protein interactions.

Amino Acid Sequence↗
PubMed1998

Glycan engineering of proteins with whole living yeast cells expressing rat liver alpha2,3-sialytransferase in the porous cell wall.

The N-glycans of recombinant proteins produced via the secretory pathway of cultured mammalian cells are often undersialylated, and insect cells lack sialytransferases. Undersialylated glycoproteins are rapidly cleared from the circulation, compromising the effect of pharmaceuticals. We show that incubation with living Saccharomyces cerevisiae cells expressing the catalytic ectodomain of rat liver alpha2,3-sialyltransferase (ST3Ne) in the porous cell wall resulted in sialylation of glycoproteins. The Km values of the yeast enzyme for several substrates were similar to those of recombinant ST3Ne from insect cells and of authentic ST3N. The yeast strain provides an inexpensive self-perpetuating source of ST3N activity for glycan engineering of recombinant proteins.

Animals↗
PubMed2005

Entropy and surface engineering in protein crystallization.

Protein crystallization remains a key limiting step in the characterization of the atomic structures of proteins and their complexes by X-ray diffraction methods. Current data indicate that standard screening procedures applied to soluble well folded prokaryotic proteins yield X-ray diffraction crystals with an approximately 20% success rate and for eukaryotic proteins this figure may be significantly lower. Protein crystallization is predominantly dependent on entropic effects and the driving force appears to be the release of ordered water from the sites of crystal contacts. This is countered by the entropic cost of ordering of protein molecules and by the loss of conformational freedom of side chains involved in the crystal contacts. Mutational surface engineering designed to create patches with low conformational entropy and thereby conducive to formation of crystal contacts promises to be an effective tool allowing direct enhancement of the success rate of macromolecular crystallization.

Crystallization↗
PubMed1994

Chemo-enzymic backbone engineering of proteins. Site-specific incorporation of synthetic peptides that mimic the 64-74 disulfide loop of granulocyte colony-stimulating factor.

We present the concept of chemo-enzymic backbone engineering of proteins. Recombinant DNA techniques are used to produce appropriate proteins that are enzymically fragmented to give the starting materials. These fragments are modified specifically at their chain termini either enzymically (coupling of a hydrazide to the C terminus) or chemically (periodate oxidation of N-terminal serine to a glyoxylyl function). The modified fragments, which need no side protection whatever, are mixed together and religate themselves spontaneously under mild conditions. The hydrazone bond thus formed can be reduced if desired, which stabilizes the linkage and enhances the flexibility of the local conformation. In this way biologically or chemically derived structures can be incorporated into the protein, and the choice of the chemical ones is free of all of the constraints of the genetic code. We believe that this combined approach gives access to constructions that could not be derived by either recombinant or chemical methods alone. We illustrate the particularity of this concept by the engineered modifications of the 64-74 disulfide loop region of human granulocyte colony-stimulating factor. Analogs constructed include one which, in spite of having a nonpeptide link in its backbone, has full biological activity.

Amino Acid Sequence↗
PubMed2001

Protein-based materials, toward a new level of structural control.

Through billions of years of evolution nature has created and refined structural proteins for a wide variety of specific purposes. Amino acid sequences and their associated folding patterns combine to create elastic, rigid or tough materials. In many respects, nature's intricately designed products provide challenging examples for materials scientists, but translation of natural structural concepts into bio-inspired materials requires a level of control of macromolecular architecture far higher than that afforded by conventional polymerization processes. An increasingly important approach to this problem has been to use biological systems for production of materials. Through protein engineering, artificial genes can be developed that encode protein-based materials with desired features. Structural elements found in nature, such as beta-sheets and alpha-helices, can be combined with great flexibility, and can be outfitted with functional elements such as cell binding sites or enzymatic domains. The possibility of incorporating non-natural amino acids increases the versatility of protein engineering still further. It is expected that such methods will have large impact in the field of materials science, and especially in biomedical materials science, in the future.

Animals↗