Construction and characterization of heterodimeric soluble quinoprotein glucose dehydrogenase

Igarashi, Satoshi; Sode, Koji

doi:10.1016/j.jbbm.2004.06.009

Cited by 7 publications

(2 citation statements)

References 22 publications

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“…In comparison, peptide tags are small, and, importantly, they do not need to be folded, which provides a significant advantage over protein tags. For example, the versatility of Arginine tags, which are used for facilitating ion exchange chromatography purification, 27,28 is well illustrated by the structure of Pyrococcus furiosus maltodextrin binding protein, which has been determined without cleavage of its Arg 5 tag sequence. 29 However, to our knowledge, poly-Arg tags have not been used for improving protein solubility, and this is the first detailed characterization of solubility enhancement both by poly-Arg and poly-Lys tags.…”

Section: Comparison With Other Tagsmentioning

confidence: 99%

Mutational analysis of protein solubility enhancement using short peptide tags

et al. 2006

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Protein aggregation is a common phenomenon. The preparation of highly concentrated protein samples, typically required for biophysical measurements, often involves a time consuming and tedious testing of solvent conditions for improving protein solubility. Here, in a systematic analysis, we have determined the increase in solubility upon the addition of SEP-tags (solubility enhancement peptide tags) containing, one, three, and five lysines or arginines (or six arginines) to either the N or C terminus of our low solubility model protein, bovine pancreatic trypsin inhibitor variant, BPTI-22 (a BPTI variant containing 22 alanines). As anticipated, the BPTI-22 solubility increased in direct relation to the number of charged residues contained in the SEP-tag, and without altering either the activity or the structure of the protein. The largest solubility increases were of 4.2-, 4.8-, and 6.2-folds produced by the addition, at the C terminus, of five lysine (BPTI-22-C5K), five and six arginine residues (BPTI-22-C5R and BPTI-22-C6R), respectively. The increased solubility of the tagged BPTI-22 yielded higher quality NMR spectra (hetero single quantum correlation HSQC spectra; with respect of the signal-to-noise and line shapes) in a much shorter time than for the untagged BPTI-22. Furthermore, tagged samples remained soluble for over ten days, as observed by their HSQC spectra. We believe that lysine- and arginine-based SEP-tags may provide an effective and versatile method for enhancing protein solubility.

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Section: Comparison With Other Tagsmentioning

confidence: 99%

Mutational analysis of protein solubility enhancement using short peptide tags

et al. 2006

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“…29 The formation of heterodimers where one subunit has been inactivated or rendered more sensitive to inhibition has been previously used to study communication between active sites of homodimers. [30][31][32] However, by modifying the substrate specificity of one of the active sites our method allows the investigator to measure allosteric effects between two active sites that are fully functional. The developed technology should be applicable to other homooligomeric enzymes for which it is possible to change the substrate specificity such as dehydrogenases 33 or tRNA synthetases.…”

Section: Allosteric Communication Between Active Sitesmentioning

confidence: 99%

Engineering homooligomeric proteins to detect weak intersite allosteric communication: Aminotransferases, a case study

Deu

Kirsch

2011

Protein Science

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The existence of low levels of intersubunit communication in homooligomeric enzymes is often difficult to discover, as the identical active sites cannot be probed individually to dissect their interdependent contributions. The homodimeric paralogs, E. coli aspartate-(AATase) and tyrosine aminotransferase (TATase), have not been demonstrated to show allostery. To address this question, we engineered a hybrid aminotransferase containing two distinct catalytic pockets: an AATase and a TATase site. The TATase/AATase hybrid was constructed by grafting an engineered TATase active site into one of the catalytic pockets of E. coli AATase. Each active site conserves its specific catalytic and inhibitor binding properties, and the hybrid catalyzes simultaneously each aminotransferase reaction at the respective site. Importantly, association of a selective inhibitor into one of the catalytic pockets decreases the activity of the second active site by up to 25%, thus proving unequivocally the existence of allosteric communication between active sites. The procedure may be applicable to other homologous sets of enzymes.

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Probing the Molecular Mechanisms in Copper Amine Oxidases by Generating Heterodimers

Gaule

Smith

Pearson³

et al. 2015

ChemBioChem

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For some homodimeric copper amine oxidases (CuAO), there is suggestive evidence of differential activity at the two active sites implying potential cooperativity between the two monomers. To examine this phenomenon for the Arthrobacter globiformis CuAO (AGAO), we purified a heterodimeric form of the enzyme for comparison with the homodimer. The heterodimer comprises an active wild-type monomer and an inactive monomer in which an active-site tyrosine is mutated to phenylalanine (Y382F). This mutation prevents the formation of the trihydroxyphenylalanine quinone (TPQ) cofactor. A pETDuet vector and a dual fusion tag strategy was used to purify heterodimers (WT/Y382F) from homodimers. Purity was confirmed by western blot and native PAGE analyses. Spectral and kinetic studies support the view that whether there are one or two functional monomers in the dimer, the properties of each functional monomer are the same, thus indicating no communication between the active sites in this bacterial enzyme.

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Construction and characterization of heterodimeric soluble quinoprotein glucose dehydrogenase

Cited by 7 publications

References 22 publications

Mutational analysis of protein solubility enhancement using short peptide tags

Mutational analysis of protein solubility enhancement using short peptide tags

Engineering homooligomeric proteins to detect weak intersite allosteric communication: Aminotransferases, a case study

Probing the Molecular Mechanisms in Copper Amine Oxidases by Generating Heterodimers

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