High-throughput protein purification and quality assessment for crystallization

Kim, Young Chang; Babnigg, G.; Jedrzejczak, R.; Eschenfeldt, William H.; Li, Hui; Maltseva, Natalia; Hatzos-Skintges, C.; Gu, M.; Makowska-Grzyska, M.; Wu, R.; An, Hao; Chhor, G.; Joachimiak, A.

doi:10.1016/j.ymeth.2011.07.010

Cited by 138 publications

(128 citation statements)

References 49 publications

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“…SeMet HcaR from Acinetobacter sp. ADP1 was purified using the procedure described earlier (37,38). The harvested cells were thawed, and 1 mg/ml lysozyme was added.…”

Section: Methodsmentioning

confidence: 99%

How Aromatic Compounds Block DNA Binding of HcaR Catabolite Regulator

Kim

Joachimiak

Bigelow

et al. 2016

Journal of Biological Chemistry

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Bacterial catabolism of aromatic compounds from various sources including phenylpropanoids and flavonoids that are abundant in soil plays an important role in the recycling of carbon in the ecosystem. We have determined the crystal structures of apo-HcaR from Acinetobacter sp. ADP1, a MarR/SlyA transcription factor, in complexes with hydroxycinnamates and a specific DNA operator. The protein regulates the expression of the hca catabolic operon in Acinetobacter and related bacterial strains, allowing utilization of hydroxycinnamates as sole sources of carbon. HcaR binds multiple ligands, and as a result the transcription of genes encoding several catabolic enzymes is increased. The 1.9 -2.4 Å resolution structures presented here explain how HcaR recognizes four ligands (ferulate, 3,4-dihydroxybenzoate, p-coumarate, and vanillin) using the same binding site. The ligand promiscuity appears to be an adaptation to match a broad specificity of hydroxycinnamate catabolic enzymes while responding to toxic thioester intermediates. Structures of apo-HcaR and in complex with a specific DNA hca operator when combined with binding studies of hydroxycinnamates show how aromatic ligands render HcaR unproductive in recognizing a specific DNA target. The current study contributes to a better understanding of the hca catabolic operon regulation mechanism by the transcription factor HcaR.

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“…SeMet HcaR from Acinetobacter sp. ADP1 was purified using the procedure described earlier (37,38). The harvested cells were thawed, and 1 mg/ml lysozyme was added.…”

Section: Methodsmentioning

confidence: 99%

How Aromatic Compounds Block DNA Binding of HcaR Catabolite Regulator

Kim

Joachimiak

Bigelow

et al. 2016

Journal of Biological Chemistry

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“…Untagged E. coli S was prepared from inclusion bodies and refolded as described previously (21). His 6 -tagged P. mirabilis Crl with Se-Met substitutions was overexpressed and purified by Ni-nitrilotriacetic acid (NTA) affinity chromatography and the AKTAxpress system (GE Health Systems), as described previously (22), and the affinity tag was cleaved with His-tagged tobacco etch virus protease, followed by an additional Ni-NTA purification to separate the protease, uncut protein, and affinity tag. E. coli His 6 -Crl was overexpressed and purified using Ni affinity and heparin chromatography, and the tag was removed by thrombin cleavage.…”

Section: Methodsmentioning

confidence: 99%

Structure of the RNA Polymerase Assembly Factor Crl and Identification of Its Interaction Surface with Sigma S

et al. 2014

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bBacteria utilize multiple sigma factors that associate with core RNA polymerase (RNAP) to control transcription in response to changes in environmental conditions. In Escherichia coli and Salmonella enterica, Crl positively regulates the S regulon by binding to S to promote its association with core RNAP. We recently characterized the determinants in S responsible for specific binding to Crl. However, little is known about the determinants in Crl required for this interaction. Here, we present the X-ray crystal structure of a Crl homolog from Proteus mirabilis in conjunction with in vivo and in vitro approaches that probe the Crl-S interaction in E. coli. We show that the P. mirabilis, Vibrio harveyi, and E. coli Crl homologs function similarly in E. coli, indicating that Crl structure and function are likely conserved throughout gammaproteobacteria. We utilize phylogenetic conservation and bacterial two-hybrid analyses to predict residues in Crl important for the interaction with S . The results of p-benzoylphenylalanine (BPA)-mediated UV cross-linking studies further support the model in which an evolutionarily conserved central cleft is the surface on Crl that binds to S . Within this conserved binding surface, we identify a key residue in Crl that is critical for activation of E S -dependent transcription in vivo and in vitro. Our study provides a physical basis for understanding the S -Crl interaction.

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“…Consequently, in many cases TEV protease (but not R3C protease) can be used to generate a digestion product with no non-native amino acid residues appended to its N-terminus. Nonetheless, it is common practice to digest fusion proteins overnight at 4°C [7,8], and it has been rumored that R3C protease has significantly greater catalytic activity at this temperature than does TEV protease [2,9]. Remarkably, however, no study has ever been carried out in which the temperature-dependence of the two enzymes has been directly compared.…”

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confidence: 99%

Differential temperature dependence of tobacco etch virus and rhinovirus 3C proteases

Raran-Kurussi

Tőzsér

Cherry

et al. 2013

Analytical Biochemistry

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Because of their stringent sequence specificity, the 3C-like proteases from tobacco etch virus (TEV3) and human rhinovirus are often used for the removal of affinity tags. The latter enzyme is rumored to have greater catalytic activity at 4°C, the temperature at which fusion protein substrates are usually digested. Here, we report that experiments with fusion protein and peptide substrates confirm this conjecture. Whereas the catalytic efficiency of rhinovirus 3C protease is approximately the same at its optimum temperature (30°C) and at 4°C, TEV protease is 10-fold less active at the latter temperature, due primarily to a reduction in kcat.

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High-throughput protein purification and quality assessment for crystallization

Cited by 138 publications

References 49 publications

How Aromatic Compounds Block DNA Binding of HcaR Catabolite Regulator

How Aromatic Compounds Block DNA Binding of HcaR Catabolite Regulator

Structure of the RNA Polymerase Assembly Factor Crl and Identification of Its Interaction Surface with Sigma S

Differential temperature dependence of tobacco etch virus and rhinovirus 3C proteases

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