An efficient method to eliminate the protease activity contaminating commercial bovine pancreatic DNase I

Le, Tien Canh; Lee, Hak Jin; Jin, Hyung Jong

doi:10.1016/j.ab.2015.04.030

Cited by 4 publications

(12 citation statements)

References 18 publications

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“…Among these, band 2 was purified using the His-tag located at the C-terminal end of the protein, separated by SDS-PAGE, extracted, and subjected to N-terminal sequencing. On the basis of the results, it was determined that the band originated from a cleavage at F67 (similar to the column numbering) . Band 1 was detected after a 1 min incubation and after Ni 2+ affinity chromatography, implying that the cleavage occurred near the N-terminus.…”

Section: Resultsmentioning

confidence: 68%

“…On the basis of the results, it was determined that the band originated from a cleavage at F67 (similar to the column numbering). 13 Band 1 was detected after a 1 min incubation and after Ni 2+ affinity chromatography, implying that the cleavage occurred near the N-terminus. Therefore, this band was thought to originate from a cleavage after Y23 (similar to the column numbering), showing a mobility similar to that of the deduced molecular weight of the cleaved fragment (34.4 kDa).…”

Section: Resultsmentioning

confidence: 95%

“…Expression and purification of the His 6 -tagged Erm proteins were conducted using a previously described strategy, with slight modifications. 13 , 37 Briefly, E. coli cells overexpressing the desired protein by culturing at 22 °C for 18–20 h after IPTG (1 mM) induction were harvested, and each 1 g of wet cell mass was resuspended in 15 mL of buffer A [500 mM KCl, 50 mM Tris-HCl (pH 7.5), and 10 mM MgCl 2 ] containing 5 mM imidazole and 1 mM PMSF. The disruption of the cells was accomplished by sonication on ice using a GEX-130 ultrasonic processor (130 W, 20 kHz) at 50% amplitude for 5 s pulses with 10 s intervals for cooling.…”

Section: Materials and Methodsmentioning

confidence: 99%

“…A previous report revealed that ErmN was seemingly more susceptible than ErmS to contaminating proteases in a commercial DNase I preparation. Accordingly, the current study aimed to elucidate the difference in proteolytic susceptibility between ErmS and ErmN and to discuss the consequences of the apparent increased susceptibility of ErmN and its effects on our daily experiments.…”

Section: Introductionmentioning

confidence: 96%

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Recognition Site Generated by Natural Changes in Erm Proteins Leads to Unexpectedly High Susceptibility to Chymotrypsin

Lee

Jin

2017

ACS Omega

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Erms are proteins that methylate the adenine (A2058) in Escherichia coli 23S rRNA, which results in resistance to macrolide, lincosamide, and streptogramin B antibiotics. In a previous report, ErmN appeared to be more susceptible to contaminating proteases in DNase I. To determine the underlying mechanism, cleavage with chymotrypsin over time was investigated. ErmN possesses unusually high-susceptibility recognition site (F45) as evidenced by a band (band 1) that represented greater than 80% of the total band intensity at 30 s. The exposure rate of the hydrophobic core was more than 67-fold and 104-fold faster in ErmN than those in ErmS and ErmE, respectively. After cleavage at F45, some of the hydrophobic interactions were disrupted. Further digestion of band 1 occurred through the exposed F163 with a half-life of 3.18 min. After 30 min, less than 1% of ErmN remained. On the basis of the structure of ErmC′, the location of F45 was presumed to be in an α helix at the bottom of a cavity. Both substitution of most common amino acids such as isoleucine, valine, or leucine with phenylalanine (ErmH, ErmI, ErmN, and ErmZ out of the 37 known Erms) and the apparent added flexibility, which could result from the additional loop region attached to phenylalanine that is four to nine amino acids longer (ErmI, ErmN, and ErmZ, which form one cluster in the phylogenetic tree), could cause unusually high susceptibility. The unexpectedly high susceptibility among the homologous proteins could indicate that caution should be taken not to misinterpret the observations when conducting any procedure in which protease or protease contamination is involved.

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Section: Resultsmentioning

confidence: 68%

Section: Resultsmentioning

confidence: 95%

Section: Materials and Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 96%

See 2 more Smart Citations

Recognition Site Generated by Natural Changes in Erm Proteins Leads to Unexpectedly High Susceptibility to Chymotrypsin

Lee

Jin

2017

ACS Omega

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“…These include organophosphate (OP) nerve agents, and both OP and carbamate insecticides [7]. Phenylmethylsulfonyl fluoride (PMSF) is a sulfonylating agent commonly employed as a non-specific inhibitor of serine proteases [8]. It was reported by Fahrney & Gold [9] that Electrophorus electricus AChE is resistant to inhibition by PMSF, 4 although it was later reported that it is indeed inhibited, but extremely slowly [10].…”

Section: Introductionmentioning

confidence: 99%

Molecular Dynamics Simulations of the Interaction of Mouse andTorpedoAcetylcholinesterase with Covalent Inhibitors Explain Their Differential Reactivity: Implications for Drug Design

Chandar

Efremenko

Silman

et al. 2019

Preprint

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Although the three-dimensional structures of mouse and Torpedo californica acetylcholinesterase are very similar, their responses to the covalent sulfonylating agents benzenesulfonyl fluoride and phenylmethylsulfonyl fluoride are qualitatively different.Both agents inhibit the mouse enzyme effectively by covalent modification of its activesite serine. In contrast, whereas the Torpedo enzyme is effectively inhibited by benzenesulfonyl fluoride, it is completely resistant to phenylmethylsulfonyl fluoride. A bottleneck midway down the active-site gorge in both enzymes restricts access of ligands to the active site at the bottom of the gorge. Molecular dynamics simulations revealed that the mouse enzyme is substantially more flexible than the Torpedo enzyme, suggesting that enhanced 'breathing motions' of the mouse enzyme relative to the Torpedo enzyme might explain why phenylmethylsulfonyl fluoride can reach the active site in mouse acetylcholinesterase, but not in the Torpedo enzyme. Accordingly, we performed docking of the two sulfonylating agents to the two enzymes, followed by molecular dynamics simulations. Whereas benzenesulfonyl fluoride closely approached the active-site serine in both mouse and Torpedo acetylcholinesterase in such simulations, phenylmethylsulfonyl 2 fluoride was able to approach the active-site serine of mouse acetylcholinesterase -but remained trapped above the bottleneck in the case of the Torpedo enzyme. Our studies demonstrate that reliance on docking tools in drug design can produce misleading information. Docking studies should, therefore, also be complemented by molecular dynamics simulations in selection of lead compounds. Author summaryEnzymes are protein molecules that catalyze chemical reactions in living organisms, and are essential for their physiological functions. Proteins have well defined threedimensional structures, but display flexibility; it is believed that this flexibility, known as their dynamics, plays a role in their function. Here we studied the neuronal enzyme acetylcholinesterase, which breaks down the neurotransmitter, acetylcholine. The active site of this enzyme is deeply buried, and accessed by a narrow gorge. A particular inhibitor, phenylmethylsulfonyl fluoride, is known to inhibit mouse acetylcholinesterase, but not that of the electric fish, Torpedo, even though their structures are very similar. A theoretical technique called molecular dynamics (MD) shows that the mouse enzyme is more flexible than the Torpedo enzyme. Furthermore, when the movement of the inhibitor down the gorge towards the active site is simulated using MD, the phenylmethylsulfonyl fluoride can reach the active site in the mouse enzyme, but not in the Torpedo enzyme, in which it remains trapped midway down the gorge. Our study emphasizes the importance of taking into account not only structure, but also dynamics, in designing drugs targeted towards proteins.

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Molecular dynamics simulations of the interaction of Mouse and Torpedo acetylcholinesterase with covalent inhibitors explain their differential reactivity: Implications for drug design

Chandar

Efremenko

Silman

et al. 2019

Chemico-Biological Interactions

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An efficient method to eliminate the protease activity contaminating commercial bovine pancreatic DNase I

Cited by 4 publications

References 18 publications

Recognition Site Generated by Natural Changes in Erm Proteins Leads to Unexpectedly High Susceptibility to Chymotrypsin

Recognition Site Generated by Natural Changes in Erm Proteins Leads to Unexpectedly High Susceptibility to Chymotrypsin

Molecular Dynamics Simulations of the Interaction of Mouse andTorpedoAcetylcholinesterase with Covalent Inhibitors Explain Their Differential Reactivity: Implications for Drug Design

Molecular dynamics simulations of the interaction of Mouse and Torpedo acetylcholinesterase with covalent inhibitors explain their differential reactivity: Implications for drug design

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