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Sökning: L773:1741 0126 OR L773:1741 0134

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1.
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4.
  • Casaite, Vida, et al. (författare)
  • Expression and purification of active recombinant equine lysozyme in Escherichia coli
  • 2009
  • Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 22:11, s. 649-654
  • Tidskriftsartikel (refereegranskat)abstract
    • Equine lysozyme (EL) is a calcium (Ca)-binding lysozyme and is an intermediary link between non-Ca-binding C-type lysozyme and alpha-lactalbumin. The feature of lysozymes to assemble into the fibrils has recently gained considerable attention for the investigation of the functional properties of these proteins. To study the structural and functional properties of EL, a synthetic gene was cloned and EL was overexpressed in Escherichia coli as a fused protein. The His-tagged recombinant EL was accumulated as inclusion bodies. Up to 50 mg/l of the recombinant EL could be achieved after purification by Ni(2+) affinity chromatography, refolding in the presence of arginine, CM-Sepharose column purification following TEV protease cleavage. The purified protein was functionally active, as determined by the lysozyme activity, proving the proper folding of protein. The purified lysozyme was used for the oligomerisation studies. The protein formed amyloid fibrils during incubation in acidic pH and elevated temperature. The recombinant EL forms two types of fibrils: ring shaped and linear, similar to the native EL.
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5.
  • Chaga, Grigoriy, et al. (författare)
  • Engineering of a metal coordinating site into human glutathione transferase M1-1 based on immobilized metal ion affinity chromatography of homologous rat enzymes
  • 1994
  • Ingår i: Protein Engineering. - : Oxford University Press (OUP). - 0269-2139 .- 1460-213X. ; 7:9, s. 1115-1119
  • Tidskriftsartikel (refereegranskat)abstract
    • Rat glutathione transferase (GST) 3-3 binds to Ni(II)-iminodiacetic acid (IDA)-agarose, whereas other GSTs that are abundant in rat liver do not bind to this immobilized metal ion affinity chromatography (IMAC) adsorbent. Rat GST 3-3 contains two superficially located amino acid residues, His84 and His85, that are suitably positioned for coordination to Ni(II)-IDA-agarose. This particular structural motif is lacking in GSTs that do not bind to the IMAC matrix. Creation of an equivalent His-His structure in the homologous human GST M1-1 by protein engineering afforded a mutant enzyme that displays affinity for Ni(II)-IDA-agarose, in contrast to the wild-type GST M1-1. The results identify a distinct site that is operational in IMAC and suggest an approach to the rational design of novel integral metal coordination sites in proteins.
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6.
  • Claes, Filip, et al. (författare)
  • Exposure of a cryptic Hsp70 binding site determines the cytotoxicity of the ALS-associated SOD1-mutant A4V
  • 2019
  • Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 32:10, s. 443-457
  • Tidskriftsartikel (refereegranskat)abstract
    • The accumulation of toxic protein aggregates is thought to play a key role in a range of degenerative pathologies, but it remains unclear why aggregation of polypeptides into non-native assemblies is toxic and why cellular clearance pathways offer ineffective protection. We here study the A4V mutant of SOD1, which forms toxic aggregates in motor neurons of patients with familial amyotrophic lateral sclerosis (ALS). A comparison of the location of aggregation prone regions (APRs) and Hsp70 binding sites in the denatured state of SOD1 reveals that ALS-associated mutations promote exposure of the APRs more than the strongest Hsc/Hsp70 binding site that we could detect. Mutations designed to increase the exposure of this Hsp70 interaction site in the denatured state promote aggregation but also display an increased interaction with Hsp70 chaperones. Depending on the cell type, in vitro this resulted in cellular inclusion body formation or increased clearance, accompanied with a suppression of cytotoxicity. The latter was also observed in a zebrafish model in vivo. Our results suggest that the uncontrolled accumulation of toxic SOD1(A4V) aggregates results from insufficient detection by the cellular surveillance network.
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7.
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8.
  • Crona, Mikael, 1981-, et al. (författare)
  • Subunit and small-molecule interaction of ribonucleotide reductases via surface plasmon resonance biosensor analyses
  • 2010
  • Ingår i: Protein Engineering Design & Selection. - : Oxford University Press. - 1741-0126 .- 1741-0134. ; 23:8, s. 633-641
  • Tidskriftsartikel (refereegranskat)abstract
    • Ribonucleotide reductase (RNR) synthesizes deoxyribonucleotides for DNA replication and repair and is controlled by sophisticated allosteric regulation involving differential affinity of nucleotides for regulatory sites. We have developed a robust and sensitive method for coupling biotinylated RNRs to surface plasmon resonance streptavidin biosensor chips via a 30.5 Å linker. In comprehensive studies on three RNRs effector nucleotides strengthened holoenzyme interactions, whereas substrate had no effect on subunit interactions. The RNRs differed in their response to the negative allosteric effector dATP that binds to an ATP-cone domain. A tight RNR complex was formed in Escherichia coli class Ia RNR with a functional ATP cone. No strengthening of subunit interactions was observed in the class Ib RNR from the human pathogen Bacillus anthracis that lacks the ATP cone. A moderate strengthening was seen in the atypical Aeromonas hydrophila phage 1 class Ia RNR that has a split catalytic subunit and a non-functional ATP cone with remnant dATP-mediated regulatory features. We also successfully immobilized a functional catalytic NrdA subunit of the E.coli enzyme, facilitating study of nucleotide interactions. Our surface plasmon resonance methodology has the potential to provide biological insight into nucleotide-mediated regulation of any RNR, and can be used for high-throughput screening of potential RNR inhibitors
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9.
  • Eldridge, Bill, et al. (författare)
  • An in vitro selection strategy for conferring protease resistance to ligand binding peptides
  • 2009
  • Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 22:11, s. 691-698
  • Tidskriftsartikel (refereegranskat)abstract
    • One drawback to the use of peptides as therapeutics has been their susceptibility to proteolysis. Here, we have used an in vitro display technology, CIS display, to enhance the proteolytic resistance of ligand-binding peptides by selection of protecting motifs from a large peptide library. The premise to this selection was that certain linear peptides within a library could form structures capable of preventing the access of proteases to defined cleavage sites without affecting ligand binding. A diverse 12-mer peptide library was inserted between a FLAG epitope motif and a thrombin cleavage site and this construct was fused to the bacterial initiator protein RepA for CIS display selection. After five rounds of selection, protection motifs were isolated that were capable of preventing proteolytic cleavage of the adjacent thrombin site. Some of the selected peptides were also resistant to more promiscuous proteases, such as chymotrypsin and trypsin, which were not used in the selection. The observed resistance to thrombin, trypsin and chymotrypsin translated into increased resistance to plasma proteases in vitro and to an increase in circulating half-lives in rats. This method can be applied to enhancing the in vivo stability of therapeutic peptides.
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10.
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11.
  • Falahati, Hanieh, et al. (författare)
  • Transmitting the allosteric signal in methylglyoxal synthase
  • 2013
  • Ingår i: Protein Engineering Design & Selection. - : Oxford University Press. - 1741-0126 .- 1741-0134. ; 26:7, s. 445-452
  • Tidskriftsartikel (refereegranskat)abstract
    • The homohexameric enzyme methylglyoxal synthase (MGS) converts dihydroxyacetone phosphate (DHAP) to methylglyoxal and phosphate. This enzyme is allosterically inhibited by phosphate. The allosteric signal induced by phosphate in MGS from Thermus sp. GH5 (TMGS) has been tracked by site-directed mutagenesis, from the binding site of phosphate to the pathways that transmit the signal, and finally to the active site which is the receiver of the signal. In TMGS, Ser-55 distinguishes the inhibitory phosphate from the phosphoryl group of the substrate, DHAP, and transmits the allosteric signal through Pro-82, Arg-97 and Val-101 to the active site. Furthermore, the addition of a C-terminal tail to TMGS reinforces the allosteric signal by introducing a new salt bridge between Asp-10 and an Arg in this tail. Lastly, the active site amino acid, Gly-56, is shown to be involved in both allostery and phosphate elimination step from DHAP by TMGS. Interestingly, some of the mutations also trigger homotropic allostery, supporting the hypothesis that allostery is an intrinsic property of all dynamic proteins. The details of the TMGS allosteric network discussed in this study can serve as a model system for understanding the enigmatic allosteric mechanism of other proteins.
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12.
  • Friedman, Mikaela, et al. (författare)
  • Phage display selection of Affibody molecules with specific binding to the extracellular domain of the epidermal growth factor receptor
  • 2007
  • Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 20:4, s. 189-199
  • Tidskriftsartikel (refereegranskat)abstract
    • Affibody molecules specific for the epidermal growth factor receptor (EGFR) have been selected by phage display technology from a combinatorial protein library based on the 58-residue, protein A-derived Z domain. EGFR is overexpressed in various malignancies and is frequently associated with poor patient prognosis, and the information provided by targeting this receptor could facilitate both patient diagnostics and treatment. Three selected Affibody variants were shown to selectively bind to the extracellular domain of EGFR (EGFR-ECD). Kinetic biosensor analysis revealed that the three monomeric Affibody molecules bound with similar affinity, ranging from 130 to 185 nM. Head-to-tail dimers of the Affibody molecules were compared for their binding to recombinant EGFR-ECD in biosensor analysis and in human epithelial cancer A431 cells. Although the dimeric Affibody variants were found to bind in a range of 25-50 nM affinities in biosensor analysis, they were found to be low nanomolar binders in the cellular assays. Competition assays using radiolabeled Affibody dimers confirmed specific EGFR-binding and demonstrated that the three Affibody molecules competed for the same epitope. Immunofluorescence microscopy demonstrated that the selected Affibody dimers were initially binding to EGFR at the cell surface of A431, and confocal microscopy analysis showed that the Affibody dimers could thereafter be internalized. The potential use of the described Affibody molecules as targeting agents for radionuclide based imaging applications in various carcinomas is discussed.
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13.
  • Gautier, Candice, et al. (författare)
  • Seeking allosteric networks in PDZ domains
  • 2018
  • Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 31:10, s. 367-373
  • Forskningsöversikt (refereegranskat)abstract
    • Ever since Ranganathan and coworkers subjected the covariation of amino acid residues in the postsynaptic density-95/Discs large/Zonula occludens 1 (PDZ) domain family to a statistical correlation analysis, PDZ domains have represented a paradigmatic family to explore single domain protein allostery. Nevertheless, several theoretical and experimental studies in the past two decades have contributed contradicting results with regard to structural localization of the allosteric networks, or even questioned their actual existence in PDZ domains. In this review, we first describe theoretical and experimental approaches that were used to probe the energetic network (s) in PDZ domains. We then compare the proposed networks for two well-studied PDZ domains namely the third PDZ domain from PSD-95 and the second PDZ domain from PTP-BL. Our analysis highlights the contradiction between the different methods and calls for additional work to better understand these allosteric phenomena.
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14.
  • Georgiev, Alexander, et al. (författare)
  • Binding specificities of the GYF domains from two Saccharomyces cerevisiae paralogs.
  • 2007
  • Ingår i: Protein Eng Des Sel. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 20:9, s. 443-52
  • Tidskriftsartikel (refereegranskat)abstract
    • We have used multivariate statistics and z-scales to represent peptide sequencesin a PLS-QSAR model of previously studied binding affinities [Kofler,M.,Motzny,K. and Freund,C. (2005b) Mol. Cell. Proteomics, 4, 1797-1811.] of two GYFdomains to an array of immobilized synthetic peptides. As a result, weestablished structural determinants of the binding specificities of the twoproteins. Our model was used to define new sets of yeast proteins potentiallyinteracting with Syh1 (YPL105C) and Smy2 (YBR172C). These sets were subsequentlyexamined for co-occurrence of Gene Ontology terms, leading to suggest a group oflikely interacting proteins with a common function in mRNA catabolism. Finally,subcellular localization of a GFP-fused Syh1 and Smy2 reinforced the possibilitythat these proteins reside in cytoplasmic sites of mRNA degradation, therebyproviding experimental confirmation to the predictions from the model.
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15.
  • Geschwindner, Stefan, et al. (författare)
  • Characterisation of de novo mutations in the C-terminal domain of proprotein convertase subtilisin/kexin type 9.
  • 2015
  • Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134.
  • Tidskriftsartikel (refereegranskat)abstract
    • Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes the degradation of the hepatic low-density lipoprotein receptor (LDL-R) and is therefore a prominent therapeutic target for reducing LDL-cholesterol. The C-terminal domain of PCSK9 is unlikely to be involved in a direct extracellular interaction with the LDL-R. We probed the importance of the C-terminus for the degradation of the LDL-R by designing seven de novo mutants of PCSK9 that fill potential druggable cavities. The mutants were tested for their ability to diminish LDL uptake in human HepG2 cells and for affinity towards a calcium independent mutant of the EGF(A) domain of the human LDL-R. The later was done by a newly developed surface plasmon resonance-based assay format. We identified three mutant proteins (G517R, V610R and V644R) with decreased ability to block LDL uptake into HepG2 cells. These mutations define areas outside the direct interaction area between PCSK9 and the LDL-R that could be targeted to inhibit the PCSK9 triggered degradation of the LDL-R. We also describe the mechanistic rationalisation of the affinity changes seen with the natural occurring human D374Y (gain of function) mutation causing severe hypercholesterolaemia. The action of this mutant is due to a significantly decreased dissociation rate constant, whereas the mutation does not affect the association rate constant.
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16.
  • Gianni, Stefano, et al. (författare)
  • Affinity versus specificity in coupled binding and folding reactions
  • 2019
  • Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 32:8, s. 355-357
  • Forskningsöversikt (refereegranskat)abstract
    • Intrinsically disordered protein regions may fold upon binding to an interaction partner. It is often argued that such coupled binding and folding enables the combination of high specificity with low affinity. The basic tenet is that an unfavorable folding equilibrium will make the overall binding weaker while maintaining the interaction interface. While theoretically solid, we argue that this concept may be misleading for intrinsically disordered proteins. In fact, experimental evidence suggests that interactions of disordered regions usually involve extended conformations. In such cases, the disordered region is exceptionally unlikely to fold into a bound conformation in the absence of its binding partner. Instead, these disordered regions can bind to their partners in multiple different conformations and then fold into the native bound complex, thus, if anything, increasing the affinity through folding. We concede that (de)stabilization of native structural elements such as helices will modulate affinity, but this could work both ways, decreasing or increasing the stability of the complex. Moreover, experimental data show that intrinsically disordered binding regions display a range of affinities and specificities dictated by the particular side chains and length of the disordered region and not necessarily by the fact that they are disordered. We find it more likely that intrinsically disordered regions are common in protein-protein interactions because they increase the repertoire of binding partners, providing an accessible route to evolve interactions rather than providing a stability-affinity trade-off.
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17.
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18.
  • Gräslund, Torbjörn, et al. (författare)
  • Charge engineering of a protein domain to allow efficient ion-exchange recovery
  • 2000
  • Ingår i: Protein Engineering. - : Oxford University Press (OUP). - 0269-2139 .- 1460-213X. ; 13:10, s. 703-709
  • Tidskriftsartikel (refereegranskat)abstract
    • We have created protein domains with extreme surface charge. These mutated domains allow for ion-exchange chromatography under conditions favourable for selective and efficient capture, using Escherichia coli as a host organism. The staphylococcal protein A-derived domain Z (Z(wt)) was used asa scaffold when constructing two mutants, Z(basic1) and Z(basic2), with high positive surface charge. Far-ultraviolet circular dichroism measurements showed that they have a secondary structure content comparable to the parental molecule Z(wt). Although melting temperatures (T-m) of the engineered domains were lower than that of the wild-type Z domain, both mutants could be produced successfully as intracellular full-length products in E. coli and purified to homogeneity by ion-exchange chromatography. Further studies performed on Z(basic1) and Z(basic2) showed that they were able to bind to a cation exchanger even at pH values in the 9 to 11 range. A gene fusion between Z(basic2) and the acidic human serum albumin binding domain (ABD), derived from streptococcal protein G, was also constructed. The gene product Z(basic2)-ABD could be purified using cation-exchange chromatography from a whole cell lysate to more than 90% purity.
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19.
  • Gulich, S., et al. (författare)
  • Engineering streptococcal protein G for increased alkaline stability
  • 2002
  • Ingår i: Protein Engineering. - : Oxford University Press (OUP). - 0269-2139 .- 1460-213X. ; 15:10, s. 835-842
  • Tidskriftsartikel (refereegranskat)abstract
    • Most protein-based affinity chromatography media are very sensitive towards alkaline treatment, which is a preferred method for regeneration and removal of contaminants from the purification devices in industrial applications. In a previous study, we concluded that a simple and straightforward strategy consisting of replacing asparagine residues could improve the stability towards alkaline conditions. In this study, we have shown the potential of this rationale by stabilizing an IgG-binding domain of streptococcal protein G, i.e. the C2 domain. In order to analyze the contribution of the different amino acids to the alkaline sensitivity of the domain we used a single point mutation strategy. Amino acids known to be susceptible towards high pH, asparagine and glutamine, were substituted for less-alkali-susceptible residues. In addition, aspartic acid residues were mutated to evaluate if the stability could be further increased. The stability of the different C2 variants was subsequently analyzed by exposing them to NaOH. The obtained results reveal that the most sensitive amino acid towards alkaline conditions in the structure of C2 is Asn36. The double mutant, C2(N7,36A), was found to be the most stable mutant constructed. In addition to the increased alkaline stability and also very important for potential use as an affinity ligand, this mutated variant also retains the secondary structure, as well as the affinity to the Fc fragment of IgG.
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20.
  • Gunnarsson, Lavinia Cicortas, 1977-, et al. (författare)
  • A carbohydrate binding module as a diversity-carrying scaffold
  • 2004
  • Ingår i: Protein Engineering Design & Selection. - : Oxford University Press. - 1741-0126 .- 1741-0134. ; 17:3, s. 213-221
  • Tidskriftsartikel (refereegranskat)abstract
    • The growing field of biotechnology is in constant need of binding proteins with novel properties. Not just binding specificities and affinities but also structural stability and productivity are important characteristics for the purpose of large-scale applications. In order to find such molecules, libraries are created by diversifying naturally occurring binding proteins, which in those cases serve as scaffolds. In this study, we investigated the use of a thermostable carbohydrate binding module, CBM4-2, from a xylanase found in Rhodothermus marinus, as a diversity-carrying scaffold. A combinatorial library was created by introducing restricted variation at 12 positions in the carbohydrate binding site of the CBM4-2. Despite the small size of the library (1.6 x 10(6) clones), variants specific towards different carbohydrate polymers (birchwood xylan, Avicel and ivory nut mannan) as well as a glycoprotein (human IgG4) were successfully selected for, using the phage display method. Investigated clones showed a high productivity (on average 69 mg of purified protein/l shake flask culture) when produced in Escherichia coli and they were all stable molecules displaying a high melting transition temperature (75.7 +/- 5.3 degrees C). All our results demonstrate that the CBM4-2 molecule is a suitable scaffold for creating variants useful in different biotechnological applications.
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21.
  • Gustafsson, Erika, et al. (författare)
  • Directed evolution of chemotaxis inhibitory protein of Staphylococcus aureus generates biologically functional variants with reduced interaction with human antibodies
  • 2010
  • Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 23:2, s. 91-101
  • Tidskriftsartikel (refereegranskat)abstract
    • Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) is a protein that binds and blocks the C5a receptor (C5aR) and formylated peptide receptor, thereby inhibiting the immune cell recruitment associated with inflammation. If CHIPS was less reactive with existing human antibodies, it would be a promising anti-inflammatory drug candidate. Therefore, we applied directed evolution and computational/rational design to the CHIPS gene in order to generate new CHIPS variants displaying lower interaction with human IgG, yet retaining biological function. The optimization was performed in four rounds: one round of random mutagenesis to add diversity into the CHIPS gene and three rounds of DNA recombination by Fragment INduced Diversity (FIND((R))). Every round was screened by phage selection and/or ELISA for decreased interaction with human IgG and retained C5aR binding. The mean binding of human anti-CHIPS IgG decreased with every round of evolution. For further optimization, new amino acid substitutions were introduced by rational design, based on the mutations identified during directed evolution. Finally, seven CHIPS variants with low interaction with human IgG and retained C5aR blocking capacity could be identified.
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22.
  • Gustavsson, M., et al. (författare)
  • Stable linker peptides for a cellulose-binding domain-lipase fusion protein expressed in Pichia pastoris
  • 2001
  • Ingår i: Protein Engineering. - : Oxford University Press (OUP). - 0269-2139 .- 1460-213X. ; 14:9, s. 711-715
  • Tidskriftsartikel (refereegranskat)abstract
    • Fusion proteins composed of a cellulose-binding domain from Neocallimastix patriciarum cellulase A and Candida antarctica lipase B were constructed using different linker peptides. The aim was to create proteolytically stable linkers that were able to join the functional modules without disrupting their function. Six fusion variants containing linkers of 4-44 residues were expressed in Pichia pastoris and analysed. Three variants were found to be stable throughout 7-day cultivations. The cellulose-binding capacities of fusion proteins containing short linkers were slightly lower compared with those containing long linkers. The lipase-specific activities of all variants, in solution or immobilized on to cellulose, were equal to that of the wildtype lipase.
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23.
  • Haloi, Nandan, et al. (författare)
  • Interactive computational and experimental approaches improve the sensitivity of periplasmic binding protein-based nicotine biosensors for measurements in biofluids
  • 2024
  • Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 37
  • Tidskriftsartikel (refereegranskat)abstract
    • We developed fluorescent protein sensors for nicotine with improved sensitivity. For iNicSnFR12 at pH 7.4, the proportionality constant for ∆F/F0 vs [nicotine] (δ-slope, 2.7 μM−1) is 6.1-fold higher than the previously reported iNicSnFR3a. The activated state of iNicSnFR12 has a fluorescence quantum yield of at least 0.6. We measured similar dose-response relations for the nicotine-induced absorbance increase and fluorescence increase, suggesting that the absorbance increase leads to the fluorescence increase via the previously described nicotine-induced conformational change, the ‘candle snuffer’ mechanism. Molecular dynamics (MD) simulations identified a binding pose for nicotine, previously indeterminate from experimental data. MD simulations also showed that Helix 4 of the periplasmic binding protein (PBP) domain appears tilted in iNicSnFR12 relative to iNicSnFR3a, likely altering allosteric network(s) that link the ligand binding site to the fluorophore. In thermal melt experiments, nicotine stabilized the PBP of the tested iNicSnFR variants. iNicSnFR12 resolved nicotine in diluted mouse and human serum at 100 nM, the peak [nicotine] that occurs during smoking or vaping, and possibly at the decreasing levels during intervals between sessions. NicSnFR12 was also partially activated by unidentified endogenous ligand(s) in biofluids. Improved iNicSnFR12 variants could become the molecular sensors in continuous nicotine monitors for animal and human biofluids.
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24.
  • Hedhammar, My, et al. (författare)
  • Negatively charged purification tags for selective anion-exchange recovery
  • 2004
  • Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 17:11, s. 779-786
  • Tidskriftsartikel (refereegranskat)abstract
    •  A novel strategy for the highly selective purification of recombinant fusion proteins using negatively charged protein domains, which were constructed by protein design, is described. A triple alpha-helical domain of 58 amino acids was used as scaffold. Far-ultraviolet circular dichroism measurements showed that the designed domains had very low alpha-helicity in a low-conductivity environment in contrast to the scaffold. The secondary structure could be induced by adding salt, giving a structure comparable to the parental molecule. Further studies showed that the new domains were able to bind to an anion exchanger even at pH values down to 5 and 6. Gene fusions between one of the designed domains and different target proteins, such as green fluorescent protein (GFP), maltose binding protein (MBP) and firefly luciferase, were also constructed. These gene products could be efficiently purified from whole cell lysates at pH 6 using anion-exchange chromatography.
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25.
  • Heinze, Birgit, et al. (författare)
  • Highly enantioselective kinetic resolution of two tertiary alcohols using mutants of an esterase from Bacillus subtilis
  • 2007
  • Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 20:3, s. 125-131
  • Tidskriftsartikel (refereegranskat)abstract
    • Enzyme-catalyzed kinetic resolutions of secondary alcohols are a standard procedure today and several lipases and esterases have been described to show high activity and enantioselectivity. In contrast, tertiary alcohols and their esters are accepted only by a few biocatalysts. Only lipases and esterases with a conserved GGG(A)X-motif are active, but show low activity combined with low enantioselectivity in the hydrolysis of tertiary alcohol esters. We show in this work that the problematic autohydrolysis of certain compounds can be overcome by medium and substrate engineering. Thus, 3-phenylbut-1-yn-3-yl acetate was hydrolyzed by the esterase from Bacillus subtilis (BS2, mutant Gly105Ala) with an enantioselectivity of E = 56 in the presence of 20% (v/v) DMSO compared to E = 28 without a cosolvent. Molecular modeling was used to study the interactions between BS2 and tertiary alcohol esters in their transition state in the active site of the enzyme. Guided by molecular modeling, enzyme variants with highly increased enantioselectivity were created. For example, a Glu188Asp mutant converted the trifluoromethyl analog of 3-phenylbut-1-yn-3-yl acetate with an excellent enantioselectivity (E > 100) yielding the (S)-alcohol with > 99%ee. In summary, protein engineering combined with medium and substrate engineering afforded tertiary alcohols of very high enantiomeric purity.
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26.
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27.
  • Holmberg, Niklas, et al. (författare)
  • Redesign of the coenzyme specificity in L-lactate dehydrogenase from bacillus stearothermophilus using site-directed mutagenesis and media engineering
  • 1999
  • Ingår i: Protein Engineering. - : Oxford University Press (OUP). - 0269-2139. ; 12:10, s. 6-851
  • Tidskriftsartikel (refereegranskat)abstract
    • L-lactate dehydrogenase (LDH) from Bacillus stearothermophilus is a redox enzyme which has a strong preference for NADH over NADPH as coenzyme. To exclude NADPH from the coenzyme-binding pocket, LDH contains a conserved aspartate residue at position 52. However, this residue is probably not solely responsible for the NADH specificity. In this report we examine the possibilities of altering the coenzyme specificity of LDH by introducing a range of different point mutations in the coenzyme-binding domain. Furthermore, after choosing the mutant with the highest selectivity for NADPH, we also investigated the possibility of further altering the coenzyme specificity by adding an organic solvent to the reaction mixture. The LDH mutant, I51K:D52S, exhibited a 56-fold increased specificity to NADPH over the wild-type LDH in a reaction mixture containing 15% methanol. Furthermore, the NADPH turnover number of this mutant was increased almost fourfold as compared with wild-type LDH. To explain the altered coenzyme specificity exhibited by the D52SI51K double mutant, molecular dynamics simulations were performed.
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28.
  • Irbäck, A, et al. (författare)
  • Binary assignments of amino acids from pattern conservation
  • 1997
  • Ingår i: Protein Engineering. - : Oxford University Press (OUP). - 0269-2139. ; 10:9, s. 1013-1017
  • Tidskriftsartikel (refereegranskat)abstract
    • We have developed a simple optimization procedure for assigning binary values to amino acids. The binary values are determined by a maximization of the degree of pattern conservation in groups of closely related protein sequences. The maximization is carried out at fixed composition. For compositions approximately corresponding to an equipartition of the residues, the optimal encoding is found to be strongly correlated with hydrophobicity. The stability of the procedure is demonstrated. Our calculations are based upon sequences in the SWISS-PROT database.
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29.
  • Ivarsson, Ylva, et al. (författare)
  • The folding pathway of an engineered circularly permuted PDZ domain
  • 2008
  • Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 21:3, s. 155-160
  • Tidskriftsartikel (refereegranskat)abstract
    • To understand the role of sequence connectivity in the folding pathway of a multi-state protein, we have analysed the folding kinetics of an engineered circularly permuted PDZ domain. This variant has been designed with the specific aim of posing two of the strands participating in the stabilisation of an early folding nucleus as contiguous elements in the primary structure. Folding of the circularly permuted PDZ2 has been explored by a variety of different experimental approaches including stopped-flow and continuous-flow kinetics, as well as ligand-induced folding experiments. Data reveal that although circular permutation introduces a significant destabilisation of the native state, a folding intermediate is stabilised and accumulated prior folding. Furthermore, quantitative analysis of the observed kinetics indicates an acceleration of the early folding events by more than two orders of magnitude. The results support the importance of sequence connectivity both in the mechanism and the speed of protein folding.
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30.
  • Jemth, Per, et al. (författare)
  • Demonstration by burst-phase analysis of a robust folding intermediate in the FF domain
  • 2008
  • Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 21:3, s. 207-214
  • Tidskriftsartikel (refereegranskat)abstract
    • The role of intermediates in the folding reaction of single-domain proteins is a controversial issue. It was previously shown by different methods that an on-pathway intermediate is populated in the presence of sodium sulphate during the folding of the FF domain from HYPA/FBP11. Here we demonstrate using analysis of the amplitudes of kinetic traces that this burst-phase folding intermediate is present at different salt concentration and at various pH, and is also found in roughly 30 site-directed mutants. The intermediate appears robust to changing conditions and thus fulfils an important criterion for a productive molecular species on the folding reaction pathway.
  •  
31.
  • Jirholt, P., et al. (författare)
  • A central core structure in an antibody variable domain determines antigen specificity
  • 2001
  • Ingår i: Protein Engineering. - : Oxford University Press (OUP). - 1460-213X. ; 14:1, s. 67-74
  • Tidskriftsartikel (refereegranskat)abstract
    • Antibody binding sites provide an adaptable surface capable of interacting with essentially any molecular target. Using CDR shuffling, residues important for the assembly of mucin-1 specific paratopes were defined by random recombination of the complementarity determining regions derived from a set of mucin-1 specific clones, previously selected from an antibody fragment library. It was found that positions 33 and 50 in the heavy chain and 32, 34, 90, 91 and 96 in the light chain were conserved in many of the clones. These particular residues seem to be located centrally in the binding site as indicated by a structure model analysis. The importance of several of these conserved residues was supported by their presence in a mouse monoclonal antibody with a known structure and the same epitope specificity. Several of these corresponding residues in the mouse monoclonal antibody are known to interact with the antigen. In conclusion, critical residues important for maintaining a human antigen-specific binding site during the process of in vitro antibody evolution were defined. Furthermore, an explanation for the observed restricted germline gene usage in certain antibody responses against protein epitopes is provided.
  •  
32.
  • Jonas, Kristina, et al. (författare)
  • Isolation of novel single-chain Cro proteins targeted for binding to the bcl-2 transcription initiation site by repertoire selection and subunit combinatorics
  • 2005
  • Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 18:11, s. 537-546
  • Tidskriftsartikel (refereegranskat)abstract
    • New designed DNA-binding proteins may be recruited to act as transcriptional regulators and could provide new therapeutic agents in the treatment of genetic disorders such as cancer. We have isolated tailored DNA-binding proteins selected for affinity to a region spanning the transcription initiation site of the human bcl-2 gene. The proteins were derived from a single-chain derivative of the lambda Cro protein (scCro), randomly mutated in its recognition helices to construct libraries of protein variants of distinct DNA-binding properties. By phage display-afforded affinity selections combined with recombination of shuffled subunits, protein variants were isolated, which displayed high affinity for the target bcl-2 sequence, as determined by electrophoretic mobility shift and biosensor assays. The proteins analyzed were moderately sequence-specific but provide a starting point for further maturation of desired function.
  •  
33.
  • Jonsson, Andreas, 1974-, et al. (författare)
  • Engineering of a femtomolar affinity binding protein to human serum albumin
  • 2008
  • Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 21:8, s. 515-527
  • Tidskriftsartikel (refereegranskat)abstract
    • We describe the development of a novel serum albumin binding protein showing an extremely high affinity (K(D)) for HSA in the femtomolar range. Using a naturally occurring 46-residue three-helix bundle albumin binding domain (ABD) of nanomolar affinity for HSA as template, 15 residues were targeted for a combinatorial protein engineering strategy to identify variants showing improved HSA affinities. Sequencing of 55 unique phage display-selected clones showed a strong bias for wild-type residues at nine positions, whereas various changes were observed at other positions, including charge shifts. Additionally, a few non-designed substitutions appeared. On the basis of the sequences of 12 variants showing high overall binding affinities and slow dissociation rate kinetics, a set of seven 'second generation' variants were constructed. One variant denoted ABD035 displaying wild-type-like secondary structure content and excellent thermal denaturation/renaturation properties showed an apparent affinity for HSA in the range of 50-500 fM, corresponding to several orders of magnitude improvement compared with the wild-type domain. The ABD035 variant also showed an improved affinity toward serum albumin from a number of other species, and a capture experiment involving human serum indicated that the selectivity for serum albumin had not been compromised from the affinity engineering.
  •  
34.
  •  
35.
  •  
36.
  • Khan, Ashhar, et al. (författare)
  • Adsorption of unfolded Cu/Zn superoxide dismutase onto hydrophobic surfaces catalyzes its formation of amyloid fibrils
  • 2019
  • Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 32:2, s. 77-85
  • Tidskriftsartikel (refereegranskat)abstract
    • Intracellular aggregates of superoxide dismutase 1 (SOD1) are associated with amyotrophic lateral sclerosis. In vivo, aggregation occurs in a complex and dense molecular environment with chemically heterogeneous surfaces. To investigate how SOD1 fibril formation is affected by surfaces, we used an in vitro model system enabling us to vary the molecular features of both SOD1 and the surfaces, as well as the surface area. We compared fibril formation in hydrophilic and hydrophobic sample wells, as a function of denaturant concentration and extraneous hydrophobic surface area. In the presence of hydrophobic surfaces, SOD1 unfolding promotes fibril nucleation. By contrast, in the presence of hydrophilic surfaces, increasing denaturant concentration retards the onset of fibril formation. We conclude that the mechanism of fibril formation depends on the surrounding surfaces and that the nucleating species might correspond to different conformational states of SOD1 depending on the nature of these surfaces.
  •  
37.
  • Kronqvist, Nina, et al. (författare)
  • A novel affinity protein selection system based on staphylococcal cell surface display and flow cytometry
  • 2008
  • Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 21:4, s. 247-255
  • Tidskriftsartikel (refereegranskat)abstract
    • Here we describe the first reported use of a Gram-positive bacterial system for the selection of affinity proteins from large combinatorial libraries displayed on the surface of Staphylococcus carnosus. An affibody library of 3 x 109 variants, based on a 58 residue domain from staphylococcal protein A, was pre-enriched for binding to human tumor necrosis factor-alpha (TNF-alpha) using one cycle of phage display and thereafter transferred to the staphylococcal host (106 variants). The staphylococcal-displayed library was subjected to three rounds of flow-cytometric sorting, and the selected clones were screened and ranked by on-cell analysis for binding to TNF-alpha and further characterized using biosensor analysis and circular dichroism spectroscopy. The successful sorting yielded three different high-affinity binders (ranging from 95 pM to 2.2 nM) and constitutes the first selection of a novel affinity protein using Gram-positive bacterial display. The method combines the simplicity of working with a bacterial host with the advantages of displaying recombinant proteins on robust Gram-positive bacteria as well as using powerful flow cytometry in the selection and characterization process.
  •  
38.
  • Kronqvist, Nina, et al. (författare)
  • Combining phage and staphylococcal surface display for generation of ErbB3-specific Affibody molecules
  • 2011
  • Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 24:4, s. 385-396
  • Tidskriftsartikel (refereegranskat)abstract
    • Emerging evidence suggests that the catalytically inactive ErbB3 (HER3) protein plays a fundamental role in normal tyrosine kinase receptor signaling as well as in aberrant functioning of these signaling pathways, resulting in several forms of human cancers. ErbB3 has recently also been implicated in resistance to ErbB2-targeting therapies. Here we report the generation of high-affinity ErbB3-specific Affibody molecules intended for future molecular imaging and biotherapeutic applications. Using a high-complexity phage-displayed Affibody library, a number of ErbB3 binders were isolated and specific cell-binding activity was demonstrated in immunofluorescence microscopic studies. Subsequently, a second-generation library was constructed based on sequences of the candidates from the phage display selection. By exploiting the sensitive affinity discrimination capacity of a novel bacterial surface display technology, the affinity of candidate Affibody molecules was further increased down to subnanomolar affinity. In summary, the demonstrated specific targeting of native ErbB3 receptor on human cancer cell lines as well as competition with the heregulin/ErbB3 interaction indicates that these novel biological agents may become useful tools for diagnostic and therapeutic targeting of ErbB3-expressing cancers. Our studies also highlight the powerful approach of combining the advantages of different display technologies for generation of functional high-affinity protein-based binders. Potential future applications, such as radionuclide-based diagnosis and treatment of human cancers are discussed.
  •  
39.
  • Kurtovic, Sanela, et al. (författare)
  • Diverging catalytic capacities and selectivity profiles with haloalkane substrates of chimeric alpha class glutathione transferases
  • 2008
  • Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 21:5, s. 329-341
  • Tidskriftsartikel (refereegranskat)abstract
    • Six homologous Alpha class glutathione transferases of human, bovine, and rat origins were hybridized by means of DNA shuffling. The chimeric mutants were compared with the parental enzymes in their activities with several alkyl iodides. In order to facilitate a multivariate analysis of relationships between substrates and enzyme activities, three descriptors were introduced: 'specific catalytic capacity', 'substrate selectivity', and 'unit-scaled substrate selectivity'. In some cases the purified mutants showed higher specific activity with a certain alkyl iodide than any of the parental enzymes. However, the overriding effect of DNA shuffling was the generation of chimeras with altered substrate selectivity profiles and catalytic capacities. The altered substrate selectivity profiles of some mutants could be rationalized by changes of the substrate-binding residues in the active site of the enzyme. However, in four of the isolated mutants all active-site residues were found identical with those of rat GST A2-2, even though their substrate specificity profiles were significantly different. Clearly, amino acid residues distant from first-sphere interactions with the substrate influence the catalytic activity. These results are relevant both to the understanding how functional properties may develop in natural enzyme evolution and in the tailoring of novel functions in protein engineering.
  •  
40.
  • Kurtovic, Sanela, et al. (författare)
  • Multivariate-activity mining for molecular quasi-species in a glutathione transferase mutant library
  • 2007
  • Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 20:5, s. 243-256
  • Tidskriftsartikel (refereegranskat)abstract
    • A library of recombinant glutathione transferases (GSTs) generated by shuffling of DNA encoding human GST M1-1 and GST M2-2 was screened with eight alternative substrates, and the activities were subjected to multivariate analysis. Assays were made in lysates of bacteria in which the GST variants had been expressed. The primary data showed clustering of the activities in eight-dimensional substrate-activity space. For an incisive analysis, the rows of the data matrix, corresponding to the different enzyme variants, were individually scaled to unit length, thus accounting for different expression levels of the enzymes. The columns representing the activities with alternative substrates were subsequently individually normalized to unit variance and a zero mean. By this standardization, the data were adjusted to comparable orders of magnitude. Three molecular quasi-species were recognized by multivariate K-means and principal component analyses. Two of them encompassed the parental GST M1-1 and GST M2-2. A third one diverged functionally by displaying enhanced activities with some substrates and suppressed activities with signature substrates for GST M1-1 and GST M2-2. A fourth cluster contained mutants with impaired functions and was not regarded as a quasi-species. Sequence analysis of representatives of the mutant clusters demonstrated that the majority of the variants in the diverging novel quasi-species were structurally similar to the M1-like GSTs, but distinguished themselves from GST M1-1 by a Ser to Thr substitution in the active site. The data show that multivariate analysis of functional profiles can identify small structural changes influencing the evolution of enzymes with novel substrate-activity profiles.
  •  
41.
  •  
42.
  • Li, He, et al. (författare)
  • The effect of thermostabilising mutations on the pressure stability of Trichoderma reesei GH11 xylanase
  • 2012
  • Ingår i: Protein Engineering, Design & Selection. - : Oxford University Press. - 1741-0134 .- 1741-0126. ; 25:12, s. 821-826
  • Tidskriftsartikel (refereegranskat)abstract
    • We studied the pressure stability of disulphide bridge mutants of Trichoderma reesei XYNII at 500–5000 bar. The inactivation of XYNII and its mutants was strongest above 4000 bar. The pressure stability correlated with the thermostability order of the XYNII mutants, indicating that the stabilising mutations in protein regions important for thermostability also protect the enzyme at high pressure. In combination with high pressure, a mild heating had already inactivated the wild-type enzyme; the thermostabilising mutations largely counteracted this effect. At a low temperature, the mutations did not have any remarkable pressure stabilisation effect. Thus, thermal inactivation appeared to dominate over pressure inactivation at higher temperatures. Kinetic calculations indicated that pressure compressibility correlated with the thermostability of xylanase mutants.
  •  
43.
  • Lindborg, M., et al. (författare)
  • High-affinity binding to staphylococcal protein A by an engineered dimeric Affibody molecule
  • 2013
  • Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 26:10, s. 635-644
  • Tidskriftsartikel (refereegranskat)abstract
    • Affibody molecules are engineered binding proteins, in which the three-helix bundle motif of the Z domain derived from protein A is used as a scaffold for sequence variation. We used phage display to select Affibody binders to staphylococcal protein A itself. The best binder, called ZpA963, binds with similar affinity and kinetics to the five homologous E, D, A, B and C domains of protein A, and to a five-domain protein A construct with an average dissociation constant, K-D, of 20 nM. The structure of ZpA963 in complex with the Z domain shows that it interacts with a surface on Z that is identical in the five protein A domains, which explains the multi-domain affinity. This property allows for high-affinity binding by dimeric Affibody molecules that simultaneously engage two protein A domains in a complex. We studied two ZpA963 dimers in which the subunits were linked by a C-terminal disulfide in a symmetric dimer or head-to-tail in a fusion protein, respectively. The dimers both bind protein A with high affinity, very slow off-rates and with saturation-dependent kinetics that can be understood in terms of dimer binding to multiple sites. The head-to-tail (ZpA963)(2)htt dimer binds with an off-rate of k(off) 5 10(6) s(1) and an estimated K-D 16 pM. The results illustrate how dimers of selected monomer binding proteins can provide an efficient route for engineering of high-affinity binders to targets that contain multiple homologous domains or repeated structural units.
  •  
44.
  • Linhult, M., et al. (författare)
  • Evaluation of different linker regions for multimerization and coupling chemistry for immobilization of a proteinaceous affinity ligand
  • 2003
  • Ingår i: Protein Engineering. - : Oxford University Press (OUP). - 0269-2139 .- 1460-213X. ; 16:12, s. 1147-1152
  • Tidskriftsartikel (refereegranskat)abstract
    • Alkaline conditions are generally preferred for sanitization of chromatography media by cleaning-in-place (CIP) protocols in industrial biopharmaceutical processes. The use of such rigorous conditions places stringent demands on the stability of ligands intended for use in affinity chromatography. Here, we describe efforts to meet these requirements for a divalent proteinaceous human serum albumin (HSA) binding ligand, denoted ABD* dimer. The ABD* dimer ligand was constructed by genetic head-to-tail linkage of two copies of the ABD* moiety, which is a monovalent and alkali-stabilized variant of one of the serum albumin-binding motifs of streptococcal protein G. Dimerization was performed to investigate whether a higher HSA-binding capacity could be obtained by ligand multimerization. We also investigated the influence on alkaline stability and HSA-binding capacity of three variants (VDANS, VDADS and GGGSG) of the inter-domain linker. Biosensor binding studies showed that divalent ligands coupled using non-directed chemistry demonstrate an increased molar HSA-binding capacity compared with monovalent ligands. In contrast, equal molar binding capacities were observed for both types of ligands when using directed ligand coupling chemistry involving the introduction and recruitment of a unique C-terminal cysteine residue. Significantly higher molar binding capacities were also detected when using the directed coupling chemistry. These results were confirmed in affinity chromatography binding capacity experiments, using resins containing thiol-coupled ligands. Interestingly, column sanitization studies involving exposure to 0.1 M NaOH solution ( pH 13) showed that of all the tested constructs, including the monovalent ligand, the divalent ligand construct containing the VDADS linker sequence was the most stable, retaining 95% of its binding capacity after 7 h of alkaline treatment.
  •  
45.
  • Mandrika, Ilona, et al. (författare)
  • Proteochemometric modelling of antibody-antigen interactions using SPOT synthesised peptide arrays
  • 2007
  • Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 20:6, s. 301-307
  • Tidskriftsartikel (refereegranskat)abstract
    • Proteochemometrics is a technology for the study of molecular recognition based on chemometric techniques. Here we applied it to analyse the amino acids and amino acid physico-chemical properties that are involved in antibodies' recognition of peptide antigens. To this end, we used a study system comprised by a diverse single chain antibody library derived from the murine mAb anti-p24 (HIV-1) antibody CB4-1, evaluated on peptide arrays manufactured by SPOT synthesis. The binding pattern obtained was correlated to physico-chemical descriptors (z-scales) of antibodies and peptides amino acids using partial least-squares projections to latent structures. Cross terms derived from antibody and antigen descriptors were included, which substantially improved the proteochemometric model. The final model was statistically highly satisfactory with a correlation coefficient R(2) = 0.73 and predictive ability Q(2) = 0.68. The physico-chemical properties of each interacting amino acid residue of both the peptides and the antibodies being essential for the antigen-antibody recognition could be retrieved from the model. The study shows for the first time the feasibility of using proteochemometrics to analyse the molecular recognition of antigens by antibodies.
  •  
46.
  • Martinelle, M, et al. (författare)
  • The role of Glu87 and Trp89 in the lid of Humicola lanuginosa lipase
  • 1996
  • Ingår i: Protein Engineering. - : Oxford University Press (OUP). - 0269-2139 .- 1460-213X. ; 9:6, s. 519-524
  • Tidskriftsartikel (refereegranskat)abstract
    • The importance of Glu87 and TRp89 in the lid of Humicola lanuginosa lipase for the hydrolytic activity at the water/lipid interface was investigated by site-directed mutagenesis. It was found that the effect on the hydrolytic activity upon the replacement of Trp89 with Phe, Leu, Gly or Glu was substrate dependent, The Trp89 mutants displayed an altered chain length specificity towards triglycerides, with a higher relative activity towards triacetin and trioctanoin compared with tributyrin, Trp89 was shown to be less important in the hydrolysis of vinyl esters compared with ethyl esters and triglycerides. An exclusive effect on the acylation reaction rate by the mutation of Trp89 was consistent with the data, It is suggested that Trp89 is important in the process of binding the acyl chain of the substrate into the active site for optimal acylation reaction rate, The Trp89Phe mutation resulted in an increased hydrolytic activity towards 2-alkylalkanoic acid esters. This is suggested to be due to reduction of unfavourable van der Waals contacts between Trp89 and the 2-substituent of the substrate, Thus, in contrast to natural substrates, Trp89 has a negative impact on the catalytic efficiency when substrates with bulky acyl chains are used, In contrast to the Trp89 mutations, the effect on the hydrolytic activity of the Glu87Ala mutation was almost substrate independent, 35-70% activity of wild-type lipase, A reduction of both the acylation and deacylation reaction was consistent with the data.
  •  
47.
  • Meijer, P J, et al. (författare)
  • Directed evolution of a type I antifreeze protein expressed in Escherichia coli with sodium chloride as selective pressure and its effect on antifreeze tolerance
  • 1996
  • Ingår i: Protein Engineering. - : Oxford University Press (OUP). - 0269-2139. ; 9:11, s. 4-1051
  • Tidskriftsartikel (refereegranskat)abstract
    • Both freezing tolerance and NaCl tolerance are improved when antifreeze proteins are expressed as fusion proteins with two domains of staphylococcal protein A (SPA) in Escherichia coli. To characterize these properties further we created a randomly mutated expression library in E. coli, based on the winter flounder antifreeze protein HPLC-8 component gene. Low-fidelity PCR products of this gene were fused to the spa gene encoding two domains of the SPA. The library was screened for enhanced NaCl tolerance and four clones were selected. The freezing tolerance of each of the selected clones was enhanced to varying extents. DNA sequencing of the isolated mutants revealed that the amphiphilic properties of the native antifreeze protein were essentially conserved. Furthermore, by studying the primary sequence of the randomly mutated clones, in comparison with the degree of freezing tolerance, we have identified clues which help in understanding the relationship between salt and freezing tolerance.
  •  
48.
  • Mejàre, M, et al. (författare)
  • Selection of cadmium specific hexapeptides and their expression as OmpA fusion proteins in Escherichia coli
  • 1998
  • Ingår i: Protein Engineering. - : Oxford University Press (OUP). - 0269-2139. ; 11:6, s. 94-489
  • Tidskriftsartikel (refereegranskat)abstract
    • In searching for novel peptides with affinity for cadmium, the phage display technique was utilized. In the selection procedure, cadmium ions were immobilized on a metal chelating Sepharose gel. The peptides selected from a hexapeptide library showed no homology to naturally occurring metallothioneins. From the phage clones selected in the biopanning process, phages with affinity for Cd-109 in free solution were identified. The peptide His-Ser-Gln-Lys-Val-Phe, which was found to exhibit the strongest relative affinity for Cd-109, was cloned into Escherichia coli as a fusion to the cell surface exposed area of the outer membrane protein OmpA. Escherichia coli cells expressing this peptide showed increased survival in growth media containing up to 1.2 mM CdCl2 when compared with cells not expressing this peptide on their surface.
  •  
49.
  • Modén, Olof, et al. (författare)
  • Mutational analysis of human glutathione transferase A2-2 identifies structural elements supporting high activity with the prodrug azathioprine
  • 2012
  • Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 25:4, s. 189-197
  • Tidskriftsartikel (refereegranskat)abstract
    • Glutathione transferase (GST) A2-2 is the human enzyme displaying the highest catalytic activity with the prodrug azathioprine (Aza). The reaction releases pharmacologically active 6-mercaptopurine by displacing the imidazole moiety from the Aza molecule. The GST-catalyzed reaction is of medical significance, since high rates of Aza activation may lead to adverse side effects in treated patients. The present study involves structureactivity relationships in GST A2-2 variants. Chimeric GSTs were previously generated by DNA shuffling and two peptide segments, one N-terminal and one C-terminal, were identified as primary determinants of Aza activity. The segments contain several residues of the substrate-binding H-site and their significance for supporting high Aza activity was investigated. Substitution of the corresponding two small regions in the low-activity human GST A3-3 or rat GST A3-3 by the human GST A2-2 segments generated chimeras with approximate to 10-fold enhanced Aza activity. The H-site residues Met208 and Leu213 in the C-terminal segment of GST A2-2 were mutated to produce a library with all possible residue combinations. At a calculated 93 library coverage, all of the 1880 mutants examined showed wild-type or decreased Aza activity, even though some retained activities with alternative substrates, further emphasizing the importance of this region for the targeted activity.
  •  
50.
  • Musdal, Yaman, et al. (författare)
  • Exploring sequence-function space of a poplar glutathione transferase using designed information-rich gene variants
  • 2017
  • Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 30:8, s. 543-549
  • Tidskriftsartikel (refereegranskat)abstract
    • Exploring the vicinity around a locus of a protein in sequence space may identify homologs with enhanced properties, which could become valuable in biotechnical and other applications. A rational approach to this pursuit is the use of 'infologs', i.e. synthetic sequences with specific substitutions capturing maximal sequence information derived from the evolutionary history of the protein family. Ninety-five such infolog genes of poplar glutathione transferase were synthesized and expressed in Escherichia coli, and the catalytic activities of the proteins determined with alternative substrates. Sequence-activity relationships derived from the infologs were used to design a second set of 47 infologs in which 90% of the members exceeded wild-type properties. Two mutants, C2 (V55I/E95D/D108E/A160V) and G5 (F13L/C70A/G122E), were further functionally characterized. The activities of the infologs with the alternative substrates 1-chloro-2,4-dinitrobenzene and phenethyl isothiocyanate, subject to different chemistries, were positively correlated, indicating that the examined mutations were affecting the overall catalytic competence without major shift in substrate discrimination. By contrast, the enhanced protein expressivity observed in many of the mutants were not similarly correlated with the activities. In conclusion, small libraries of well-defined infologs can be used to systematically explore sequence space to optimize proteins in multidimensional functional space.
  •  
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