Protocols for production of selenomethionine-labeled proteins in 2-L polyethylene terephthalate bottles using auto-induction medium

Sreenath, Hassan K.; Bingman, C.A.; Buchan, Blake W.; Seder, Kory D.; Burns, Brendan T.; Geetha, Holalkere V.; Jeon, Won Bae; Vojtik, Frank C.; Aceti, David J.; Frederick, Ronnie O.; Phillips, George N.; Fox, Brian G.

doi:10.1016/j.pep.2004.12.022

Cited by 105 publications

(94 citation statements)

References 27 publications

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“…The cell lysates were analyzed for total expression and the presence of Rv3230c in the soluble fraction by Western blotting with primary mouse anti-His-tagged antibody (Novagen) and SDS-PAGE. Samples were prepared and fractionated for SDS-PAGE using approaches developed for high-throughput structural genomics studies (30). For the immunoprecipitation, the lysate was treated with antibody and then the antigen-antibody complex was precipitated by addition of Protein G-linked Sepharose resin (Amersham, Piscataway, NJ).…”

Section: Fractionation Of Rv3230cmentioning

confidence: 99%

Identification of Rv3230c as the NADPH Oxidoreductase of a Two-Protein DesA3 Acyl-CoA Desaturase inMycobacterium tuberculosisH37Rv

Chen

Fox

2006

Biochemistry

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DesA3 is a membrane-bound stearoyl-CoA Δ9 desaturase that produces oleic acid, a precursor of mycobacterial membrane phospholipids and triglycerides. DesA3 has sequence homology with other membrane desaturases including the presence of the eight His motif proposed to bind the diiron center active site. This family of desaturases function as multi-component complexes, and thus require electron transfer proteins for efficient catalytic turnover. Here we present evidence that Rv3230c from Mycobacterium tuberculosis H37Rv is a biologically relevant electron transfer partner for DesA3 from the same pathogen. For these studies, Rv3230c was expressed as a partially soluble protein in Escherichia coli; recombinant DesA3 was expressed in Mycobacterium smegmatis as a catalytically active membrane protein. The addition of E. coli lysates containing Rv3230c to lysates of M. smegmatis expressing DesA3 gave strong conversion of [1-14 C]-18:0-CoA to [1-14 C]-cis-Δ 9 -18:1-CoA and of [1-14 C]-16:0-CoA to [1-14 C]-cis-Δ 9 -16:1-CoA. Both M. tuberculosis proteins were required to reconstitute activity, as various combinations of control lysates lacking either Rv3230c or DesA3 gave minimal or no activity. Furthermore, the specificity of interaction between Rv3230c and DesA3 was implied by the inability of other related redox systems to substitute for Rv3230c. The reconstituted activity was dependent upon the presence of NADPH, could be saturated by increasing the amount of Rv3230c added and was also sensitive to the salt concentration in the buffer. The results are consistent with the formation of a protein-protein complex, possibly with electrostatic character. This work defines a multi-protein, acyl-CoA desaturase complex from M. tuberculosis H37Rv to minimally consist of a soluble Rv3230c reductase and integral membrane DesA3 desaturase. Further implications of this finding relative to the properties of other multi-protein iron enzyme complexes are discussed.Mycobacterium tuberculosis is a human pathogen that causes one of the world's deadliest diseases, tuberculosis (TB 1 ). Current estimates are that up to one-third of the world's population may be infected with M. tuberculosis and that over two million people die from TB-related diseases each year (1-4). TB has resurged, in part due to the appearance of multipledrug-resistant strains. Consequently, the development of new drugs and the reexamination of drugs formerly deemed effective have become major focuses of world-wide TB research (3)(4)(5). This intense effort has included genome sequencing (6), proteome functional analysis by micro-array approaches (7-11), the TB Protein Structure Initiative [(12), * To whom correspondence should be addressed: Department of Biochemistry, 433 Babcock Drive, Madison, WI 53706. Telephone: (608) . E-mail: bgfox@biochem.wisc.edu. † This work was supported by the National Institutes of Health Grant R01 GM-50853 to B.G.F.1 Abbreviations: AlkB, alkane ω-hydroxylase; DesA3, product of Mycobacterium tuberculosis H37Rv gene rv3229c, a...

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Section: Fractionation Of Rv3230cmentioning

confidence: 99%

Identification of Rv3230c as the NADPH Oxidoreductase of a Two-Protein DesA3 Acyl-CoA Desaturase inMycobacterium tuberculosisH37Rv

Chen

Fox

2006

Biochemistry

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“…For the six proteins successfully purified, the average yield was 39 mg per liter of culture. Such high-yields and incorporation can be obtained as well in similar production media employing autoinduction and incorporating isotopically labeled amino acids for nuclear magnetic resonance experiments [23][24][25]. In addition to the application described here, highly enriched defined media and vectors of the general configuration, tag1-site1-tag2-site2-protein, could be exploited for other purposes, including functional analyses of proteins.…”

Section: Discussionmentioning

confidence: 98%

“…Cultures were grown in 2-liter polyethylene terephthalate beverage bottles [20,21] containing one liter of nonsterile M9 salts supplemented with glucose, glycerol, amino acids, trace metals and vitamins to increase the cell yield [22][23][24][25]. Amendments were, per liter: glycerol, 5 g; glucose, 4.4 g; Cultures were grown at 37 °C to an OD 600 1 -2, when inhibitory amino acids (25 mg each of L-valine, L-isoleucine, L-leucine, L-lysine, L-threonine, L-phenylalanine, and 15 mg of selenomethionine; Medicillin, Catalog No.…”

Section: Production Of Selenomethionyl Proteins In Non-sterile Enrichmentioning

confidence: 99%

“…The medium and conditions were identical to those described previously [21] but with the addition of glycerol, non-inhibitory amino acids, trace metals and vitamins [18,[22][23][24][25] to improve the yield of cells. Under these conditions, cell yields were two-to three-fold higher compared to those attained in unsupplemented medium, typically generating OD 600 values of 4-12, depending on the protein expressed, with no detriment to expression or in vivo cleavage.…”

Section: Production Of Selenomethionyl Proteins From Pmcsg19mentioning

confidence: 99%

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An expression vector tailored for large-scale, high-throughput purification of recombinant proteins

Donnelly

Zhou

Millard

et al. 2006

Protein Expression and Purification

161

172

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Production of milligram quantities of numerous proteins for structural and functional studies requires an efficient purification pipeline. We found that the dual tag, his 6 -tag-maltose-binding protein (MBP), intended to facilitate purification and enhance proteins' solubility, disrupted such a pipeline, requiring additional screening and purification steps. Not all proteins rendered soluble by fusion to MBP remained soluble after its proteolytic removal, and in those cases where the protein remained soluble, standard purification protocols failed to remove completely the stoichiometric amount of his 6 -tagged MBP generated by proteolysis. Both liabilities were alleviated by construction of a vector that produces fusion proteins in which MBP, the his 6 -tag and the target protein are separated by highly specific protease cleavage sites in the configuration MBP-site-his 6 -site-protein. In vivo cleavage at the first site by co-expressed protease generated untagged MBP and his 6 -tagged target protein.Proteins not truly rendered soluble by transient association with MBP precipitated, and untagged MBP was easily separated from the his-tagged target protein by conventional protocols. The second protease cleavage site allowed removal of the his 6 -tag. KeywordsHigh-throughput; Structural genomics; Maltose-binding protein; TVMV protease; Ligationindependent cloningThe burgeoning genomic information now available makes vast numbers of proteins accessible for structural and functional studies, and many large-scale projects have developed automated protocols for amplifying, cloning, and expressing genes, and for screening proteins for desirable properties [1][2][3][4][5]. Similar strides have been made in streamlining protein purification, but production of sufficient material for detailed structural and functional characterization remains labor-intensive and time-consuming [3,4,6,7]. Typically, purification is facilitated by fusing proteins to affinity tags, most commonly a his-tag, which allows purification by immobilized metal-ion affinity chromatography (IMAC,[8]). Additional tags are often attached to improve proteins' solubility, such as maltose-binding protein (MBP) [2][3][4]9,10]. In typical protein production pipelines, the resulting fusion proteins are first screened for ☆ This manuscript has been created by the University of Chicago as operator of Argonne National Laboratory under Contract No.W-31-109-ENG-38 with the US Department of Energy. The US government retains for itself, and others acting on its behalf, a paid-up, nonexclusive, irrevocable worldwide license in said article to reproduce, prepare derivative works, distribute copies to the public, and perform publicly and display publicly, by or on behalf of the government. The US Government's right to retain a nonexclusive royaltyfree license in and to the copyright covering this paper, for governmental purposes, is acknowledged. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript solubility, then purified by semi-robotic p...

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“…Both constructs were subcloned into pET22b, overexpressed in E. coli as C-terminal His-tagged proteins, and purified by nickel-affinity chromatography followed by size-exclusion chromatography according to Kang et al (30). SeMet-substituted C2 was produced according to a protocol from Sreenath et al (31) and purified similar to the native protein. Full details of protein expression and purification are given in SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

Autocatalytically generated Thr-Gln ester bond cross-links stabilize the repetitive Ig-domain shaft of a bacterial cell surface adhesin

Kwon

Squire

Young

et al. 2013

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

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Gram-positive bacteria are decorated by a variety of proteins that are anchored to the cell wall and project from it to mediate colonization, attachment to host cells, and pathogenesis. These proteins, and protein assemblies, such as pili, are typically long and thin yet must withstand high levels of mechanical stress and proteolytic attack. The recent discovery of intramolecular isopeptide bond cross-links, formed autocatalytically, in the pili from Streptococcus pyogenes has highlighted the role that such crosslinks can play in stabilizing such structures. We have investigated a putative cell-surface adhesin from Clostridium perfringens comprising an N-terminal adhesin domain followed by 11 repeat domains. The crystal structure of a two-domain fragment shows that each domain has an IgG-like fold and contains an unprecedented ester bond joining Thr and Gln side chains. MS confirms the presence of these bonds. We show that the bonds form through an autocatalytic intramolecular reaction catalyzed by an adjacent His residue in a serine protease-like mechanism. Two buried acidic residues assist in the reaction. By mutagenesis, we show that loss of the ester bond reduces the thermal stability drastically and increases susceptibility to proteolysis. As in pilin domains, the bonds are placed at a strategic position joining the first and last strands, even though the Ig fold type differs. Bioinformatic analysis suggests that similar domains and ester bond cross-links are widespread in Gram-positive bacterial adhesins. intramolecular ester bond | protein stability A striking feature of globular proteins is that despite the chemical diversity inherent in the side chains of their constituent amino acids, chemical reactions between these side chains are very rare. This may be explained by evolutionary selection, which minimizes reactions that could prejudice proper protein folding. Thus, the only common example of a covalent cross-link between protein side chains is the disulfide bond, which forms only in an appropriate redox environment when two Cys residues are brought together by protein folding. Nevertheless, some surprising examples of unexpected crosslinks have been brought to light by protein structure analysis or by the observation of unusual spectroscopic or biophysical properties. Examples include the Cys-Tyr bond in galactose oxidase (1), which provides a radical center; similar bonds in some catalases (2); the His-Tyr bond in cytochrome C oxidase (3); and the remarkable chromophore of GFP (4). These, and other examples, arise through intramolecular reactions facilitated by particular local environments.The recent discovery of isopeptide bonds joining Lys and Asn side chains in the proteins that make up pili on the Gram-positive bacterium Streptococcus pyogenes (5), as well as on other Gram-positive pathogens (6), has highlighted a class of proteins in which intramolecular cross-links seem to be remarkably prevalent. It includes not only Gram-positive pili but a number of other cell surface adhesins, known as mi...

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Protocols for production of selenomethionine-labeled proteins in 2-L polyethylene terephthalate bottles using auto-induction medium

Cited by 105 publications

References 27 publications

Identification of Rv3230c as the NADPH Oxidoreductase of a Two-Protein DesA3 Acyl-CoA Desaturase inMycobacterium tuberculosisH37Rv

Identification of Rv3230c as the NADPH Oxidoreductase of a Two-Protein DesA3 Acyl-CoA Desaturase inMycobacterium tuberculosisH37Rv

An expression vector tailored for large-scale, high-throughput purification of recombinant proteins

Autocatalytically generated Thr-Gln ester bond cross-links stabilize the repetitive Ig-domain shaft of a bacterial cell surface adhesin

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