High Throughput Protein Production and Crystallization at NYSGXRC

Sauder, Michael J.; Rutter, M.; Bain, K.T.; Rooney, Isabelle; Gheyi, T.; Atwell, S.; Thompson, Devon A.; Emtage, Spencer; Burley, S.K.

doi:10.1007/978-1-60327-058-8_37

Cited by 34 publications

(36 citation statements)

References 7 publications

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“…The purified sample was loaded onto a HiLoad S200 16/60 PR gel filtration column that was equilibrated with SECB buffer [20 mM Hepes (pH 7.5), 150 mM NaCl, 10% (vol/vol) glycerol, and 5 mM DTT]. Peak fractions were collected and protein was analyzed by SDS/PAGE, snap-frozen in liquid nitrogen, and stored at −80°C (48,49).…”

Section: Methodsmentioning

confidence: 99%

Panoramic view of a superfamily of phosphatases through substrate profiling

Huang

Pandya

Liu

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

136

139

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Large-scale activity profiling of enzyme superfamilies provides information about cellular functions as well as the intrinsic binding capabilities of conserved folds. Herein, the functional space of the ubiquitous haloalkanoate dehalogenase superfamily (HADSF) was revealed by screening a customized substrate library against >200 enzymes from representative prokaryotic species, enabling inferred annotation of ∼35% of the HADSF. An extremely high level of substrate ambiguity was revealed, with the majority of HADSF enzymes using more than five substrates. Substrate profiling allowed assignment of function to previously unannotated enzymes with known structure, uncovered potential new pathways, and identified isofunctional orthologs from evolutionarily distant taxonomic groups. Intriguingly, the HADSF subfamily having the least structural elaboration of the Rossmann fold catalytic domain was the most specific, consistent with the concept that domain insertions drive the evolution of new functions and that the broad specificity observed in HADSF may be a relic of this process.evolution | specificity | phosphatase | substrate screen | promiscuity S ince the first genomes were sequenced, there has been an exponential increase in the number of protein sequences deposited into databases worldwide. At the time of this writing the UniProtKB/TrEMBL database contains over 32 million protein sequences. Although this increase in sequence data has dramatically enhanced our understanding of the genomic organization of organisms, as the number of protein sequences grows, the proportion of firm functional assignments diminishes. Traditionally, methods of functional annotation involve comparing sequence identity between experimentally characterized proteins and newly sequenced ones, typically via BLAST (1). In cases where significant sequence similarity cannot be ascertained, proteins are annotated as "hypothetical" or "putative." Moreover, the decrease in sequence identity leads to an increased uncertainty in functional assignment, especially as the phylogenetic distance between organisms grows, limiting iso-functional ortholog discovery.As the number of newly sequenced genomes grows larger, more protein sequences are likely to be misannotated, oftentimes resulting in the propagation of incorrect functional annotation across newly identified sequences. To tackle the problem of unannotated or misannotated proteins, newer methods for computational assignment have been created with varying degrees of success (2). Although these methods outperform historical methods, continued improvement is necessary to ensure accurate annotation of function (2). A greater swath of functional space can be covered by screening substrates in a high-throughput manner on multiple enzymes from a family (3, 4). Family-wide substrate profiling offers a data-rich resource. The use of sparse screening of sequence space and a diversified library permits the determination of substrate specificity profiles to provide a familywide view of the range of substrates...

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Section: Methodsmentioning

confidence: 99%

Panoramic view of a superfamily of phosphatases through substrate profiling

Huang

Pandya

Liu

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

136

139

View full text Add to dashboard Cite

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“…Recombinant bacterial expression plasmids pMRLB.38, encoding the protein MPT51 (Rv3803c), pMRLB.8, encoding (36). The cloned construct demonstrating the most robust expression was selected for scaled-up production (37). The vectors were expressed in Escherichia coli, and the recombinant proteins were purified as described previously (18).…”

Section: Methodsmentioning

confidence: 99%

Antibodies against Mycobacterial Proteins as Biomarkers for HIV-Associated Smear-Negative Tuberculosis

Siev

Wilson

Kainth

et al. 2014

Clin Vaccine Immunol

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؊ (0% and 6%, respectively), and at moderate risk for TB development, i.e., U.S. HIV ؉ TST ؉ controls (14% and 10%, respectively). By contrast, more reactivity was detected in the S.A. HIV ؉ control group at higher risk for TB (25% and 45%, respectively). Our data hold promise that antibody detection against MPT51 and echA1 might have adjunctive value in the detection of HIV ؉ smear-negative TB and might reflect increasing Mycobacterium tuberculosis infection activity in asymptomatic HIV ؉ individuals.

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“…Highthroughput protein production for members of the DUF1537 family was performed (25). We produced 22 of 122 target proteins (cloned from an in-house collection of ∼400 gDNAs); at least one purified protein was obtained for most of the major clusters in the DUF1537 SSN shown in Fig.…”

Section: Synergistic Analysis Of Ssns and Gnns Enables The Predictionmentioning

confidence: 99%

Assignment of function to a domain of unknown function: DUF1537 is a new kinase family in catabolic pathways for acid sugars

Zhang

Carter

Vetting

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Using a large-scale "genomic enzymology" approach, we (i) assigned novel ATP-dependent four-carbon acid sugar kinase functions to members of the DUF1537 protein family (domain of unknown function; Pfam families PF07005 and PF17042) and (ii) discovered novel catabolic pathways for D-threonate, L-threonate, and D-erythronate. The experimentally determined ligand specificities of several solute binding proteins (SBPs) for TRAP (tripartite ATP-independent permease) transporters for four-carbon acids, including D-erythronate and L-erythronate, were used to constrain the substrates for the catabolic pathways that degrade the SBP ligands to intermediates in central carbon metabolism. Sequence similarity networks and genome neighborhood networks were used to identify the enzyme components of the pathways. Conserved genome neighborhoods encoded SBPs as well as permease components of the TRAP transporters, members of the DUF1537 family, and a member of the 4-hydroxy-L-threonine 4-phosphate dehydrogenase (PdxA) oxidative decarboxylase, class II aldolase, or ribulose 1,5-bisphosphate carboxylase/oxygenase, large subunit (RuBisCO) superfamily. Because the characterized substrates of members of the PdxA, class II aldolase, and RuBisCO superfamilies are phosphorylated, we postulated that the members of the DUF1537 family are novel ATP-dependent kinases that participate in catabolic pathways for four-carbon acid sugars. We determined that (i) the DUF1537/PdxA pair participates in a pathway for the conversion of D-threonate to dihydroxyacetone phosphate and CO 2 and (ii) the DUF1537/class II aldolase pair participates in pathways for the conversion of D-erythronate and L-threonate (epimers at carbon-3) to dihydroxyacetone phosphate and CO 2 . The physiological importance of these pathways was demonstrated in vivo by phenotypic and genetic analyses.DUF1537 | kinase | four-carbon acid sugars | conserved genome neighborhoods | genomic enzymology

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High Throughput Protein Production and Crystallization at NYSGXRC

Cited by 34 publications

References 7 publications

Panoramic view of a superfamily of phosphatases through substrate profiling

Panoramic view of a superfamily of phosphatases through substrate profiling

Antibodies against Mycobacterial Proteins as Biomarkers for HIV-Associated Smear-Negative Tuberculosis

Assignment of function to a domain of unknown function: DUF1537 is a new kinase family in catabolic pathways for acid sugars

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