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 343 records · Page 19Linked to original sources
PubMedDate not supplied

Reversible hydrogels from self-assembling genetically engineered protein block copolymers.

A series of triblock protein copolymers composed of a central water-soluble polyelectrolyte segment flanked by two coiled-coil domains was synthesized by genetic engineering methods. The copolymers self-assembled into reversible hydrogels in response to changes in temperature, pH, and the presence or absence of denaturating agent (guanidine hydrochloride, GdnHCl). Hydrogel formation was concentration-dependent, and the concentration needed for hydrogel formation correlated with the oligomerization state of the coiled-coil domains in the protein copolymers. The morphology of the hydrogels, as determined by scanning electron microscopy (SEM), indicated the presence of porous interconnected networks. The thermal stabilities and self-assembling properties of the protein copolymers were successfully controlled by manipulating the amino acid sequences of the coiled-coil domains. The stimuli responsiveness and reversibility of the hydrogel self-assembly suggest that these protein copolymers may have potential in biomedical applications.

Amino Acid Sequence↗
PubMed2007

M13 bacteriophage coat proteins engineered for improved phage display.

This chapter describes a method for increasing levels of protein fusions displayed on the surfaces of M13 bacteriophage particles. By introducing mutations into the anchoring M13 coat protein, protein display levels can be increased by up to two orders of magnitude. Experimental methods are presented for the design, construction, and screening of phage-displayed libraries for improved protein display.

Capsid Proteins↗
PubMedDate not supplied

Protein engineering of cytochrome c by semisynthesis: substitutions at glutamic acid 66.

We have used protein semisynthesis to prepare four analogues of horse cytochrome c, in which the glutamic acid residue at position 66 has been removed and replaced by norvaline, glutamine, lysine and, as a methodological control, glutamic acid. This residue is quite strongly conserved in mitochondrial cytochrome c, and forms part of a cluster of acidic residues that occurs in all cytochromes c but whose function is obscure. Comparative studies of the physical and biochemical properties of the analogues have now disclosed two specific roles for Glu66 in the protein. It contributes significantly to the stabilization of the active conformation of the protein, probably by salt bridge formation, and it appears to participate in the redox-state-dependent ATP-binding site of cytochrome c. Our results also support two general views of the role of surface charged residues in cytochrome c, namely that their disposition influences both redox potential, through the electrostatic field felt at the redox centre, and the kinetics of electron transfer, through the dipole moment they generate.

Amino Acid Sequence↗
PubMed1995

[Study of the role of histidine residues in the function of uridine phosphorylase from Escherichia coli K-12 by protein engineering].

Using site-directed mutagenesis, mutant genes of the E.coli UDPase that coded proteins with point substitutions of histidine residues (i.e., H8N, H47N, H101N, H122N, H152N, H179N, and H240N) were constructed. Study of the enzymatic activity of mutant UDPases showed that histidine-asparagine substitutions at the positions 47, 101, 152, 179, and 240 do not affect protein functioning. Whereas H122N and H8N substitutions inhibit the activity of UDPase by 60 and 100%, respectively. This evidences the important functional role of the His122 and His8 residues for the formation of the active site fo the enzyme.

Base Sequence↗
PubMed1996

Protein engineering and design for drug delivery.

Over the past decade considerable effort has been focused on the genetic construction, expression, and characterization of fusion proteins designed to selectively target specific disease-causing cells and/or extracellular targets. In each instance, the coding sequences for the functional domains of different proteins have been genetically fused, resulting in the formation of fusion proteins which retain the function of their component segments. As anticipated from their design, many of these fusion proteins offer a marked therapeutic advantage over conventional agents in preclinical studies. While several recombinant fusion proteins have entered or will shortly begin Phase I/II human clinical trials, the first of these fusion proteins is currently being evaluated in Phase III human clinical trials for the treatment of cutaneous T cell lymphoma.

Amino Acid Sequence↗
PubMed1996

Elucidation of a MgATP signal transduction pathway in the nitrogenase iron protein: formation of a conformation resembling the MgATP-bound state by protein engineering.

The present work defines one MgATP signal transduction pathway in the nitrogenase iron (Fe) protein. Deletion of an amino acid (Leu 127) by site-directed mutagenesis in the protein chain between Asp 125, located in the ATP binding site, and Cys 132, a ligand to the [4Fe-4S] cluster, resulted in protein conformational changes resembling the MgATP-bound state in the absence of any bound nucleotides. Specifically, 1H nuclear magnetic resonance, electron paramagnetic resonance, and circular dichroism spectroscopic properties, along with Fe chelation assays, suggested that deletion of Leu 127 in the Fe protein resulted in changes in the electronic properties of the [4Fe-4S] cluster similar to those normally observed upon MgATP binding to the wild-type Fe protein. Deletion of Leu 127 of the Fe protein lowered the redox potential of of the [4FE-4S] cluster by 112 mV compared to the wild-typeFe protein (-412mV compared to -294 mV). A nearly identical lowering of the redox potential by 120 mV occurs in the wild-type Fe protein upon binding MgATP (-294 mV compared to 420 mV). The L127delta Fe protein did not contain bound nucleotides which could account for the observed conformational changes. The present results support a model in which the protein chain from ASP 125 to Cys 132 acts as one pathway for MgATP signal transduction and suggests a mechanism for this transduction to the [4Fe-4S] cluster. The L127delta Fe protein was found to still bind 2 MgATP or 2 MgADP molecules/Fe protein. Unlike the wild-type Fe protein, the L127delta Fe protein bound 2 ADP molecules/Fe protein in the absence of Mg2+. Finally, the L127delta protein was found to bind to the MoFe protein, although the complex did not catalyze MgATP hydrolysis or substrate reduction. In concurrence with previous models, homologies between the Asp 125 to Cys 132 transduction pathway in Fe protein and the switch II region of the broad class of GTPase signal transduction proteins (G-proteins) are discussed.

Adenosine Triphosphate↗
PubMed2001

Rapid generation of incremental truncation libraries for protein engineering using alpha-phosphothioate nucleotides.

Incremental truncation for the creation of hybrid enzymes (ITCHY) is a novel tool for the generation of combinatorial libraries of hybrid proteins independent of DNA sequence homology. We herein report a fundamentally different methodology for creating incremental truncation libraries using nucleotide triphosphate analogs. Central to the method is the polymerase catalyzed, low frequency, random incorporation of alpha-phosphothioate dNTPs into the region of DNA targeted for truncation. The resulting phosphothioate internucleotide linkages are resistant to 3'-->5' exonuclease hydrolysis, rendering the target DNA resistant to degradation in a subsequent exonuclease III treatment. From an experimental perspective the protocol reported here to create incremental truncation libraries is simpler and less time consuming than previous approaches by combining the two gene fragments in a single vector and eliminating additional purification steps. As proof of principle, an incremental truncation library of fusions between the N-terminal fragment of Escherichia coli glycinamide ribonucleotide formyltransferase (PurN) and the C-terminal fragment of human glycinamide ribonucleotide formyltransferase (hGART) was prepared and successfully tested for functional hybrids in an auxotrophic E.coli host strain. Multiple active hybrid enzymes were identified, including ones fused in regions of low sequence homology.

Amino Acid Sequence↗
PubMed2002

Engineering protein and cell adhesivity using PEO-terminated triblock polymers.

Previous studies on customizing cell culture environments have utilized a variety of microfabrication-based tools to control the spatial localization of adhesive proteins and subsequently mammalian cells. Others have used various methods to immobilize nonadhesive PEO-based polymers on surfaces to inhibit protein absorption and cell adhesion. In this study, we report the application of a well-characterized, commercially available, PEO-terminated triblock polymer (Pluronic F108) to create micropatterned nonadhesive domains on a variety of biomaterials that deter cell adhesion for up to 4 weeks in culture. The Pluronic can be applied using microfluidic tools or photolithographic techniques, and can be adsorbed to a variety of common surfaces including tissue culture polystyrene, methylated glass, silicone, and polylactic-co-glycolic acid. The effectiveness of the Pluronic in inhibiting cell adhesion in the presence of collagen I is also quantified. Finally, these patterning techniques are generalized to control tissue organization on a variety of common biomaterials. This simple method for micropatterning PEO and, therefore, proteins and cells should prove useful as a tool for biomolecular surface engineering.

3T3 Cells↗