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 811 records · Page 45Linked to original sources
PubMed1998

Effects of metal binding affinity on the chemical and thermal stability of site-directed mutants of rat oncomodulin.

Tryptophan fluorescence was used to study the stability and unfolding behavior of several single tryptophan mutants of the metal-binding protein rat oncomodulin (OM); F102W, Y57W, Y65W and the engineered protein CDOM33 which had the 12 residues of the CD loop replaced with a more potent metal binding site. Both the thermal and the chemical stability were improved upon binding of metal ions with the order apo < Ca2+ < Tb3+. During thermal denaturation, the transition midpoints (T(un)) of Y65W was the lowest, followed by Y57W and F102W. The placement of the Trp residue in the F-helix in F102W made the protein slightly more thermostable, although the fluorescence response was readily affected by chemical denaturants, which acted through the disruption of hydrogen bonds at the C-terminal end of the F-helix. Under both thermal and chemical denaturation, the engineered protein showed the highest stability. This indicated that increasing the number of metal ligating oxygens in the binding site, either by using a metal ion with a higher coordinate number (i.e., Tb3+) which binds more carboxylate ligands, or by providing more ligating groups, as in the CDOM33 replacement, produces notable improvements in protein stability.

Acrylamide↗
PubMed2006

Engineering isoflavone metabolism with an artificial bifunctional enzyme.

Plant secondary metabolism has been a focus of research in recent years due to its significant roles in plant defense and in human medicine and nutrition. A protein engineering strategy was designed to more effectively manipulate plant secondary metabolite (isoflavonoid) biosynthesis. A bifunctional isoflavone synthase/chalcone isomerase (IFS/CHI) enzyme was constructed by in-frame gene fusion, and expressed in yeast and tobacco. The fusion protein was targeted to the endoplasmic reticulum (ER) membrane and the individual enzymatic functions of its component fragments were retained when assayed in yeast. Petals and young leaves of IFS/CHI transgenic tobacco plants produced higher levels of the isoflavone genistein and genistein glycosides as a ratio of total flavonoids produced than did plants transformed with IFS alone. Thus, through a combined molecular modeling, in vitro protein engineering and in planta metabolic engineering approach, it was possible to increase the potential for accumulation of isoflavonoid compounds in non-legume plants. Construction of bifunctional enzymes will simplify the transformation of plants with multiple pathway genes, and such enzymes may find broad uses for enzyme (e.g., cytochrome P450 family) and biochemical pathway engineering.

Artificial Gene Fusion↗
PubMed1996

A humanized system for pharmacologic control of gene expression.

Gene therapy was originally conceived as a medical intervention to replace or correct defective genes in patients with inherited disorders. However, it may have much broader potential as an alternative delivery platform for protein therapeutics, such as cytokines, hormones, antibodies and novel engineered proteins. One key technical barrier to the widespread implementation of this form of therapy is the need for precise control over the level of protein production. A suitable system for pharmacologic control of therapeutic gene expression would permit precise titration of gene product dosage, intermittent or pulsatile treatment, and ready termination of therapy by withdrawal of the activating drug. We set out to design such a system with the following properties: (1) low baseline expression and high induction ratio; (2) positive control by an orally bioavailable small-molecule drug; (3) reduced potential for immune recognition through the exclusive use of human proteins; and (4) modularity to allow the independent optimization of each component using the tools of protein engineering. We report here the properties of this system and demonstrate its use to control circulating levels of human growth hormone in mice implanted with engineered human cells.

Animals↗
PubMed2003

A fluorescent cassette-based strategy for engineering multiple domain fusion proteins.

BACKGROUND: The engineering of fusion proteins has become increasingly important and most recently has formed the basis of many biosensors, protein purification systems, and classes of new drugs. Currently, most fusion proteins consist of three or fewer domains, however, more sophisticated designs could easily involve three or more domains. Using traditional subcloning strategies, this requires micromanagement of restriction enzymes sites that results in complex workaround solutions, if any at all. RESULTS: Therefore, to aid in the efficient construction of fusion proteins involving multiple domains, we have created a new expression vector that allows us to rapidly generate a library of cassettes. Cassettes have a standard vector structure based on four specific restriction endonuclease sites and using a subtle property of blunt or compatible cohesive end restriction enzymes, they can be fused in any order and number of times. Furthermore, the insertion of PCR products into our expression vector or the recombination of cassettes can be dramatically simplified by screening for the presence or absence of fluorescence. CONCLUSIONS: Finally, the utility of this new strategy was demonstrated by the creation of basic cassettes for protein targeting to subcellular organelles and for protein purification using multiple affinity tags.

Cloning, Molecular↗
PubMed1990

Large scale protein separations: engineering aspects of chromatography.

The engineering considerations common to large scale chromatographic purification of proteins are reviewed. A discussion of the industrial chromatography fundamentals is followed by aspects which affect the scale of separation. The separation column geometry, the effect of the main operational parameters on separation performance, and the physical characteristics of column packing are treated. Throughout, the emphasis is on ion exchange and size exclusion techniques which together constitute the major portion of commercial chromatographic protein purifications. In all cases, the state of current technology is examined and areas in need of further development are noted. The physico-chemical advances now underway in chromatographic separation of biopolymers would ensure a substantially enhanced role for these techniques in industrial production of products of new biotechnology.

Journal Article↗
PubMed1995

Theoretical approaches to designing novel sequences to fit a given fold.

One of the major goals of molecular biology is to understand how protein chains fold into a unique three-dimensional structure. Given this knowledge, perhaps the most exciting prospect will be the possibility of designing new proteins to perform designated tasks. The eventual pinnacle of protein engineering will be the fully automated design of a protein with novel structure and function. Achievement of this aim lies far in the future, although some early progress has been made recently.

Amino Acid Sequence↗
PubMed1998

Redesigning small molecule-protein interfaces.

Several recent reports have described the rational redesign of small molecule-protein interfaces, generating modified ligands that interact only with suitably mutated proteins. These studies provide powerful reagents for specifically manipulating engineered proteins inside cells, as well as general insights into the factors underlying the binding affinity and specificity of small molecules in general. Progress this year includes the development of allele-specific inhibitors of Src-family tyrosine kinases and the crystal structure determination of a remodeled FKBP-ligand interface.

Adenosine Triphosphate↗
PubMed2001

Contribution of engineered electrostatic interactions to the stability of cytosolic malate dehydrogenase.

Protein engineering is a promising tool to obtain stable proteins. Comparison between homologous thermophilic and mesophilic enzymes from a given structural family can reveal structural features responsible for the enhanced stability of thermophilic proteins. Structures from pig heart cytosolic and Thermus flavus malate dehydrogenases (cMDH, Tf MDH), two proteins showing a 55% sequence homology, were compared with the aim of increasing cMDH stability using features from the Thermus flavus enzyme. Three potential salt bridges from Tf MDH were selected on the basis of their location in the protein (surface R176-D200, inter-subunit E57-K168 and intrasubunit R149-E275) and implemented on cMDH using site-directed mutagenesis. Mutants containing E275 were not produced in any detectable amount, which shows that the energy penalty of introducing a charge imbalance in a region that was not exposed to solvent was too unfavourable to allow proper folding of the protein. The salt bridge R149-E275, if formed, would not enhance stability enough to overcome this effect. The remaining mutants were expressed and active and no differences from wild-type other than stability were found. Of the mutants assayed, Q57E/L168K led to a stability increase of 0.4 kcal/mol, as determined by either guanidinium chloride denaturalization or thermal inactivation experiments. This results in a 15 degrees C shift in the optimal temperature, thus confirming that the inter-subunit salt bridge initially present in the T.flavus enzyme was formed in the cMDH structure and that the extra energy obtained is transformed into an increase in protein stability. These results indicate that the use of structural features of thermophilic enzymes, revealed by a detailed comparison of three-dimensional structures, is a valid strategy to improve the stability of mesophilic malate dehydrogenases.

Catalysis↗
PubMed1999

The study of protein mechanics with the atomic force microscope.

The unfolding and folding of single protein molecules can be studied with an atomic force microscope (AFM). Many proteins with mechanical functions contain multiple, individually folded domains with similar structures. Protein engineering techniques have enabled the construction and expression of recombinant proteins that contain multiple copies of identical domains. Thus, the AFM in combination with protein engineering has enabled the kinetic analysis of the force-induced unfolding and refolding of individual domains as well as the study of the determinants of mechanical stability.

Entropy↗
PubMed2026

SARS-CoV-2 Orf3a protein interaction mapping using unnatural amino acid incorporation.

Mapping transient protein-protein interactions remain a major challenge in studying viral host-pathogen interfaces. While some virus-host interactions are stable and readily captured, the majority are highly dynamic, reflecting the need for viral proteins to engage distinct host factors at different stages of the life cycle. Here, we employ a protein engineering strategy based on the site-specific incorporation of the unnatural acid p-azido-L-phenylalanine (AzF) to enable photo-crosslinking proteomic analysis of the SARS-CoV-2 accessory protein Orf3a in live cells. Genetic installation of AzF at residue K198 of Orf3a permitted UV-induced covalent capture of proximal host interacting proteins, overcoming challenges associated with membrane localization and limited protein abundance. A total of 248 high-confidence Orf3a-interacting proteins were reproducibly identified and subjected to gene ontology analysis, revealing enrichment in innate immune signaling, antiviral defense, RNA processing, and viral replication-associated pathways. Orf3a is an accessory protein that functions as a viroporin and traffics across multiple cellular compartments, and was found to interact with host RNA helicases, RNA-binding proteins, immune regulators, and metabolic enzymes implicated in SARS-CoV-2 infection. Together, these results demonstrate that genetically encoded, site-specific photo-crosslinking enables selective capture of transient interactions that are often missed by nonspecific 254&#xa0;nm UV crosslinking approaches and highlights Orf3a as a multifunctional protein that engages diverse host pathways. More broadly, this study establishes a generalizable framework for leveraging unnatural amino acid-based protein engineering approaches to interrogate dynamic host-pathogen interactions.

Humans↗
PubMed2005

Strategies and computational tools for improving randomized protein libraries.

In the last decade, directed evolution has become a routine approach for engineering proteins with novel or altered properties. Concurrently, a trend away from purely 'blind' randomization strategies and towards more 'semi-rational' approaches has also become apparent. In this review, we discuss ways in which structural information and predictive computational tools are playing an increasingly important role in guiding the design of randomized libraries: web servers such as ConSurf-HSSP and SCHEMA allow the prediction of sites to target for producing functional variants, while algorithms such as GLUE, PEDEL and DRIVeR are useful for estimating library completeness and diversity. In addition, we review recent methodological developments that facilitate the construction of unbiased libraries, which are inherently more diverse than biased libraries and therefore more likely to yield improved variants.

Algorithms↗
PubMed1991

Design of site-specific protein drugs.

The ability to clone and express proteins at high levels of abundance and purity has led to their increasing consideration as therapeutic agents. The use of various protein engineering and recombinant DNA techniques has enabled progression to where proteins can be tailored so that they have not only the ability to interact with a unique (extra)cellular component in order to produce a pharmacological effect, but their structure can be altered so as to achieve a selective biological disposition. This ability to control the biological dispersion of proteins is becoming a key element in the design of therapeutic polypeptides and proteins, finding application in modalities intended for treating HIV infections, accessible tumor masses, and diseases of the hematopoietic system, etc. For recombinant site-specific systems to be successfully introduced into the clinic, radically different approaches are required during their development and clinical testing. These relate not only to the pharmacokinetics and receptor availability of the drug, but also its pharmacodynamics, its analytics and its safety pharmacology. This contribution examines how the control of the biological disposition of proteins may be achieved using both site-directed mutagenesis, sequence re-ordering, hybrid protein formation, protein resurfacing, as well as synthetic adduction to polymers.

Chromosome Deletion↗
PubMed2004

Primary structure elements of spider dragline silks and their contribution to protein solubility.

Spider silk proteins have mainly been investigated with regard to their contribution to mechanical properties of the silk thread. However, little is known about the molecular mechanisms of silk assembly. As a first step toward characterizing this process, we aimed to identify primary structure elements of the garden spider's (Araneus diadematus) major dragline silk proteins ADF-3 and ADF-4 that determine protein solubility. In addition, we investigated the influence of conditions involved in mediating natural thread assembly on protein aggregation. Genes encoding spider silk-like proteins were generated using a cloning strategy, which is based on a combination of synthetic DNA modules and PCR-amplified authentic gene sequences. Comparing secondary structure, solubility, and aggregation properties of the synthesized proteins revealed that single primary structure elements have diverse influences on protein characteristics. Repetitive regions representing the largest part of dragline silk proteins determined the solubility of the synthetic proteins, which differed greatly between constructs derived from ADF-3 and ADF-4. Factors, such as acidification and increases in phosphate concentration, which promote silk assembly in vivo generally decreased silk protein solubility in vitro. Strikingly, this effect was pronounced in engineered proteins comprising the carboxyl-terminal nonrepetitive regions of ADF-3 or ADF-4, indicating that these regions might play an important role in initiating assembly of spider silk proteins.

Amino Acid Motifs↗
PubMed2004

Circular permutation as a tool to reduce surface entropy triggers crystallization of the signal recognition particle receptor beta subunit.

The production of diffraction-quality crystals remains a difficult obstacle on the road to high-resolution structural characterization of proteins. This is primarily a result of the empirical nature of the process. Although crystallization is not predictable, factors inhibiting it are well established. First, crystal formation is always entropically unfavorable. Reducing the entropic cost of crystallizing a given protein is thus desirable. It is common practice to map boundaries and remove unstructured regions surrounding the folded protein domain. However, a problem arises when flexible regions are not at the boundaries but within a domain. Such regions cannot be deleted without adding new restraints to the domain. We encountered this problem during an attempt to crystallize the beta subunit of the eukaryotic signal recognition particle (SRbeta), bearing a long and flexible internal loop. Native SRbeta did not crystallize. However, after circularly permuting the protein by connecting the spatially close N and C termini with a short heptapeptide linker GGGSGGG and removing 26 highly flexible loop residues within the domain, we obtained diffraction-quality crystals. This protein-engineering method is simple and should be applicable to other proteins, especially because N and C termini of protein domains are often close in space. The success of this method profits from prior knowledge of the domain fold, which is becoming increasingly common in today's postgenomic era.

Crystallization↗
PubMed2006

The contribution of the residues from the main hydrophobic core of ribonuclease A to its pressure-folding transition state.

The role of hydrophobic interactions established by the residues that belong to the main hydrophobic core of ribonuclease A in its pressure-folding transition state was investigated using the Phi-value method. The folding kinetics was studied using pressure-jump techniques both in the pressurization and depressurization directions. The ratio between the folding activation volume and the reaction volume (beta p-value), which is an index of the compactness or degree of solvation of the transition state, was calculated. All the positions analyzed presented fractional Phi f-values, and the lowest were those corresponding to the most critical positions for the ribonuclease A stability. The structure of the transition state of the hydrophobic core of ribonuclease A, from the point of view of formed interactions, is a relatively, uniformly expanded form of the folded structure with a mean Phi f-value of 0.43. This places it halfway between the folded and unfolded states. On the other hand, for the variants, the average of beta p-values is 0.4, suggesting a transition state that is 40% native-like. Altogether the results suggest that the pressure-folding transition state of ribonuclease A looks like a collapsed globule with some secondary structure and a weakened hydrophobic core. A good correlation was found between the Phi f-values and the Deltabeta p-values. Although the nature of the transition state inferred from pressure-induced folding studies and the results of the protein engineering method have been reported to be consistent for other proteins, to the best of our knowledge this is the first direct comparison using a set of mutants.

Amino Acid Sequence↗
PubMed2006

Inhibition of platelet aggregation by grafting RGD and KGD sequences on the structural scaffold of small disulfide-rich proteins.

Disintegrins represent a group of disulfide-rich peptides ranging in size from 41 to over 80 residues and are antagonists of several integrin receptors. Disintegrins containing an RGD or KGD sequence are potent inhibitors of platelet aggregation as they block the binding of fibrinogen to alpha(IIb)beta(3) integrin. The high affinity binding to alpha(IIb)beta(3) in comparison to short linear peptides has been attributed to the localisation of the RGD or KGD sequence within a defined three-dimensional structure. Cystine knot microproteins are members of another family of small disulfide-rich peptides that consist of only 28-40 amino acid residues. They display numerous biological activities depending on the peptide sequence of loop regions that are fixed on a structural scaffold that is stabilised by three knot-forming disulfide bonds. In the present study we grafted RGD and KGD containing peptide sequences with seven and 11 amino acids, respectively, into two cystine knot microproteins, the trypsin inhibitor EETI-II and the melanocortin receptor binding domain of the human agouti-related protein AGRP, as well as into the small disintegrin obtustatin. The engineered proteins were much more potent to inhibit the fibrinogen binding, alpha(IIb)beta(3) activation and platelet aggregation when compared to the grafted peptides. Differences that were observed between the engineered proteins indicate the importance of the structural scaffold and the amino acids neighbouring the grafted peptide sequences.

Amino Acid Sequence↗