| 1. |
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| 2. |
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| 3. |
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| 4. |
- Casaite, Vida, et al.
(author)
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Expression and purification of active recombinant equine lysozyme in Escherichia coli
- 2009
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In: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 22:11, s. 649-654
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Journal article (peer-reviewed)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.
(author)
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Engineering of a metal coordinating site into human glutathione transferase M1-1 based on immobilized metal ion affinity chromatography of homologous rat enzymes
- 1994
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In: Protein Engineering. - : Oxford University Press (OUP). - 0269-2139 .- 1460-213X. ; 7:9, s. 1115-1119
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Journal article (peer-reviewed)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.
(author)
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Exposure of a cryptic Hsp70 binding site determines the cytotoxicity of the ALS-associated SOD1-mutant A4V
- 2019
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In: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 32:10, s. 443-457
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Journal article (peer-reviewed)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.
(author)
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Subunit and small-molecule interaction of ribonucleotide reductases via surface plasmon resonance biosensor analyses
- 2010
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In: Protein Engineering Design & Selection. - : Oxford University Press. - 1741-0126 .- 1741-0134. ; 23:8, s. 633-641
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Journal article (peer-reviewed)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.
(author)
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An in vitro selection strategy for conferring protease resistance to ligand binding peptides
- 2009
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In: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 22:11, s. 691-698
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Journal article (peer-reviewed)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.
(author)
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Transmitting the allosteric signal in methylglyoxal synthase
- 2013
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In: Protein Engineering Design & Selection. - : Oxford University Press. - 1741-0126 .- 1741-0134. ; 26:7, s. 445-452
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Journal article (peer-reviewed)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.
(author)
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Phage display selection of Affibody molecules with specific binding to the extracellular domain of the epidermal growth factor receptor
- 2007
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In: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 20:4, s. 189-199
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Journal article (peer-reviewed)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.
(author)
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Seeking allosteric networks in PDZ domains
- 2018
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In: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 31:10, s. 367-373
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Research review (peer-reviewed)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.
(author)
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Binding specificities of the GYF domains from two Saccharomyces cerevisiae paralogs.
- 2007
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In: Protein Eng Des Sel. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 20:9, s. 443-52
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Journal article (peer-reviewed)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.
(author)
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Characterisation of de novo mutations in the C-terminal domain of proprotein convertase subtilisin/kexin type 9.
- 2015
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In: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134.
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Journal article (peer-reviewed)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.
(author)
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Affinity versus specificity in coupled binding and folding reactions
- 2019
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In: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 32:8, s. 355-357
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Research review (peer-reviewed)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.
(author)
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Charge engineering of a protein domain to allow efficient ion-exchange recovery
- 2000
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In: Protein Engineering. - : Oxford University Press (OUP). - 0269-2139 .- 1460-213X. ; 13:10, s. 703-709
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Journal article (peer-reviewed)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.
(author)
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Engineering streptococcal protein G for increased alkaline stability
- 2002
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In: Protein Engineering. - : Oxford University Press (OUP). - 0269-2139 .- 1460-213X. ; 15:10, s. 835-842
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Journal article (peer-reviewed)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.
(author)
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A carbohydrate binding module as a diversity-carrying scaffold
- 2004
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In: Protein Engineering Design & Selection. - : Oxford University Press. - 1741-0126 .- 1741-0134. ; 17:3, s. 213-221
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Journal article (peer-reviewed)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.
(author)
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Directed evolution of chemotaxis inhibitory protein of Staphylococcus aureus generates biologically functional variants with reduced interaction with human antibodies
- 2010
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In: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 23:2, s. 91-101
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Journal article (peer-reviewed)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.
(author)
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Stable linker peptides for a cellulose-binding domain-lipase fusion protein expressed in Pichia pastoris
- 2001
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In: Protein Engineering. - : Oxford University Press (OUP). - 0269-2139 .- 1460-213X. ; 14:9, s. 711-715
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Journal article (peer-reviewed)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.
(author)
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Interactive computational and experimental approaches improve the sensitivity of periplasmic binding protein-based nicotine biosensors for measurements in biofluids
- 2024
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In: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 37
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Journal article (peer-reviewed)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.
(author)
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Negatively charged purification tags for selective anion-exchange recovery
- 2004
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In: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 17:11, s. 779-786
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Journal article (peer-reviewed)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.
(author)
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Highly enantioselective kinetic resolution of two tertiary alcohols using mutants of an esterase from Bacillus subtilis
- 2007
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In: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 20:3, s. 125-131
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Journal article (peer-reviewed)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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