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- Casaite, Vida, et al.
(författare)
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Expression and purification of active recombinant equine lysozyme in Escherichia coli
- 2009
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 22:11, s. 649-654
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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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- Chaga, Grigoriy, et al.
(författare)
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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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Ingår i: Protein Engineering. - : Oxford University Press (OUP). - 0269-2139 .- 1460-213X. ; 7:9, s. 1115-1119
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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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- Claes, Filip, et al.
(författare)
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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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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 32:10, s. 443-457
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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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- Crona, Mikael, 1981-, et al.
(författare)
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Subunit and small-molecule interaction of ribonucleotide reductases via surface plasmon resonance biosensor analyses
- 2010
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press. - 1741-0126 .- 1741-0134. ; 23:8, s. 633-641
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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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- Eldridge, Bill, et al.
(författare)
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An in vitro selection strategy for conferring protease resistance to ligand binding peptides
- 2009
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 22:11, s. 691-698
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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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