Expression, purification, and solubility optimization of peptidyl-tRNA hydrolase 1 from Bacillus cereus

Taylor-Creel, Kasey; Hames, Mary C.; Holloway, W. Blake; McFeeters, Hana; McFeeters, Robert L.

doi:10.1016/j.pep.2014.01.007

Cited by 6 publications

(6 citation statements)

References 31 publications

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“…Having this methodology in place enables continued, higher throughput screening for antimicrobials [14,26,27]. It also provides a means for establishing Pth activity, accelerating understanding of the many emerging bacterial Pth1s [17,19,[28][29][30][31] and Pth enzymes in multi-component eukaryotic Pth systems [7,9]. Inhibition (%) Figure 5: Natural production inhibition of Pth1.…”

Section: Resultsmentioning

confidence: 99%

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A Highly Adaptable Method for Quantification of Peptidyl-tRNA Hydrolase Activity

Holloway¹,

McFeeters²,

Powell³

et al. 2015

J Anal Bioanal Tech

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The emerging importance of Peptidyl-tRNA hydrolase (Pth) enzymes necessitates the need for a widely applicable functional assay to further studies of this important enzyme family. Previously reported methods for monitoring Pth function suffer from limitations of cost, time, substrate availability, and application compatibility. Herein we present a new method for the rapid and precise characterization of Pth activity. The method is applicable for use with specific or bulk peptidyl-tRNA, any Pth enzyme, and a range of reaction conditions including solvent additives. The method also allows for semi-automated quantitative assessment of peptidyl-tRNA cleavage. No specialized equipment, harmful reagents, or time-consuming techniques are required. We use the new method to characterize Pth activity, determine enzyme kinetic parameters, screen for inhibitors, and determine inhibitory parameters.

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Section: Resultsmentioning

confidence: 99%

“…Bacterial Pth1 enzymes were expressed and purified as previously reported [17][18][19]. Human pth2 was commercially synthesized (GenScript, Piscataway, NJ) and cloned into the pET28b vector (Novagen/EMD Millipore, Billerica, MA) using NcoI and BamHI restriction sites.…”

Section: Pth Expression and Purificationmentioning

confidence: 99%

A Highly Adaptable Method for Quantification of Peptidyl-tRNA Hydrolase Activity

Holloway¹,

McFeeters²,

Powell³

et al. 2015

J Anal Bioanal Tech

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“…StPth1 was expressed and purified as previously reported [23]. Briefly, StPth1 cloned into the pKQV4 vector was transformed into BL21 E. coli cells.…”

Section: Methodsmentioning

confidence: 99%

“…The enzymatic activity of StPth1 was determined as previously published [24,25] with peptidyl-tRNA produced as before [15,23]. Briefly, acid urea minigels were utilized to determine Pth1 activity level from the difference in electrophoretic mobility of heterogeneous peptidyl-tRNA (substrate) and free tRNA (product).…”

Section: Methodsmentioning

confidence: 99%

Expedited isolation of natural product peptidyl-tRNA hydrolase inhibitors from a Pth1 affinity column

Sethi

Osier

Burks

et al. 2017

AIMS Molecular Science

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New antibiotics and new antibiotic targets are needed to counter the development of bacterial drug resistance that threatens to return the human population to the pre-antibiotic era. Bacterial peptidyl-tRNA hydrolase (Pth1) is a promising new antibiotic target in the early stages of development. While inhibitory activity has been observed in a variety of natural products, bioactive fractionation has been a bottleneck for inhibitor isolation. To expedite the isolation of inhibitory compounds from complex mixtures, we constructed a Pth1 affinity column and used it to isolate inhibitory compounds from crude natural products. Recombinantly produced S. typhimurium Pth1 was covalently attached to a column matrix and the inhibitory activity isolated from ethanol extracts of Salvinia minima. The procedure reported here demonstrates that isolation of Pth1 inhibitory compounds from complex natural product extracts can be greatly expedited over traditional bioactive fractionation, decreasing time and expense. The approach is generally applicable to Pth1s from other bacterial species and opens an avenue to advance and accelerate inhibitor development against this promising antimicrobial target.

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“…After 16 hours the cells were harvested by centrifugation and stored at -80 °C until purification. Pth1 was purified as previously described (Hughes et al 2012; McFeeters et al 2012; Taylor-Creel et al 2014). …”

Section: Methodsmentioning

confidence: 99%

Bacterial production of site specific 13C labeled phenylalanine and methodology for high level incorporation into bacterially expressed recombinant proteins

et al. 2016

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Nuclear magnetic resonance spectroscopy studies of ever larger systems have benefited from many different forms of isotope labeling, in particular, site specific isotopic labeling. Site specific 13C labeling of methyl groups has become an established means of probing systems not amenable to traditional methodology. However useful, methyl reporter sites can be limited in number and/or location. Therefore, new complementary site specific isotope labeling strategies are valuable. Aromatic amino acids make excellent probes since they are often found at important interaction interfaces and play significant structural roles. Aromatic side chains have many of the same advantages as methyl containing amino acids including distinct 13C chemical shifts and multiple magnetically equivalent 1H positions. Herein we report economical bacterial production and one-step purification of phenylalanine with 13C incorporation at the Cα, Cγ and Cε positions, resulting in two isolated 1H-13C spin systems. We also present methodology to maximize incorporation of phenylalanine into recombinantly overexpressed proteins in bacteria and demonstrate compatibility with ILV-methyl labeling. Inexpensive, site specific isotope labeled phenylalanine adds another dimension to biomolecular NMR, opening new avenues of study.

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Expression, purification, and solubility optimization of peptidyl-tRNA hydrolase 1 from Bacillus cereus

Cited by 6 publications

References 31 publications

A Highly Adaptable Method for Quantification of Peptidyl-tRNA Hydrolase Activity

A Highly Adaptable Method for Quantification of Peptidyl-tRNA Hydrolase Activity

Expedited isolation of natural product peptidyl-tRNA hydrolase inhibitors from a Pth1 affinity column

Bacterial production of site specific 13C labeled phenylalanine and methodology for high level incorporation into bacterially expressed recombinant proteins

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