Auto-induction medium for the production of [U-15N]- and [U-13C, U-15N]-labeled proteins for NMR screening and structure determination

Tyler, Robert C.; Sreenath, Hassan K.; Singh, Shanteri; Aceti, David J.; Bingman, C.A.; Markley, John L.; Fox, Brian G.

doi:10.1016/j.pep.2004.12.024

Cited by 93 publications

(72 citation statements)

References 30 publications

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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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Section: Discussionmentioning

confidence: 98%

Section: Production Of Selenomethionyl Proteins In Non-sterile Enrichmentioning

confidence: 99%

Section: Production Of Selenomethionyl Proteins From Pmcsg19mentioning

confidence: 99%

See 1 more Smart Citation

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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“…To produce 15 N myristoylated NCS-1 the ZYP-5052 media was replaced with minimal P-5052 autoinduction media as previously described [22]. 15 N supplementation of the P-5052 media was achieved with the addition of 15 NH 4 Cl obtained from Cambridge Isotopes.…”

Section: Expression Of 15 N Myristoylated Ncs-1 and Nmr Spectroscopymentioning

confidence: 99%

Optimized expression and purification of myristoylated human neuronal calcium sensor 1 in E. coli

Cotiis

Woll

Fox

et al. 2008

Protein Expression and Purification

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We have developed a protocol to produce large quantities of high purity myristoylated and nonmyristoylated Neuronal Calcium Sensor 1 (NCS-1) protein. NCS-1 is a member of the neuronal calcium sensor (NCS) family and plays an important role in modulating G-protein signaling and exocytosis pathways in cells. Many of these functions are calcium dependent and require NCS-1 to be modified with an N-terminal myristoyl moiety. In our system, a C-terminally 6X His-tagged variant of NCS-1 was co-expressed with yeast N-myristoyltransferase (NMT) in ZYP-5052 auto-induction media supplemented with sodium myristate (100 -200 µM). With optimized growth conditions and a high capacity metal affinity purification scheme, > 50 mg of homogenous myristoylated NCS-1 is obtained from 1 L of culture in a single step. The properties of the C-terminally tagged NCS-1 variants are indistinguishable from those reported for untagged NCS-1. Using this system, we have also isolated and characterized mutant NCS-1 proteins that have attenuated (NCS-1 E120Q) and abrogated (NCS-1 EF) ability to bind calcium. The large quantities of NCS-1 proteins isolated from small culture volumes of auto-inducible media will provide the necessary reagents for further biochemical and structural characterization. The affinity tag at the C-terminus of the protein provides a suitable reagent for easily identifying binding partners of the various NCS-1 constructs. Additionally, this method could be used to produce other recombinant proteins of the NCS family, and may be extended to express and isolate myristoylated variants of other proteins.

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“…[U- 13 C, 15 N]-protein for NMR investigations was produced by modifying the auto-induction growth components to include isotopically labeled compounds, 15 NH 4 Cl, [U- 13 C]-glucose and [U- 13 C]-glycerol, as the nitrogen and carbon sources. The details of the procedure have been described elsewhere (9).…”

Section: Experimental Procedures Expression and Protein Purificationmentioning

confidence: 99%

Structure of Arabidopsis thaliana At1g77540 Protein, a Minimal Acetyltransferase from the COG2388 Family^,

et al. 2006

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We describe X-ray crystal and NMR solution structures of the protein coded for by Arabidopsis thaliana gene At1g77540.1 (At1g77540). The crystal structure was determined to 1.15 Å with an R factor of 14.9% (R free = 17.0%) by multiple-wavelength anomalous diffraction using sodium bromide derivatized crystals. The ensemble of NMR conformers was determined with protein samples labeled with 15 N and 13 C+ 15 N. The X-ray structure and NMR ensemble were closely similar with r.m.s.d 1.4 Å for residues 8-93. At1g77540 was found to adopt a fold similar to that of GCN5-related N-acetyltransferases. Enzymatic activity assays established that At1g77540 possesses weak acetyltransferase activity against histones H3 and H4. Chemical shift perturbations observed in 15 N-HSQC spectra upon the addition of CoA indicated that the cofactor binds and identified its binding site. The molecular details of this interaction were further elucidated by solving the X-ray structure of the At1g77540-CoA complex. This work establishes that the domain family COG2388 represents a novel class of acetyltransferase and provides insight into possible mechanistic roles of the conserved Cys 76 and His 41 residues of this family.As part of an ongoing effort to determine the three-dimensional structures of novel eukaryotic proteins, the Center for Eukaryotic Structural Genomics (CESG 1 ) chose the gene product of † This work was supported by NIH grants P50 GM64598 and U54 GM074901 from the National Institute of General Medical Sciences (J.L.M., P.I). J.M.D. and C.E.B. acknowledge financial support from NIH grant GM059785. Use of the Advanced Photon Source and the Argonne National Laboratory Structural Biology Center beamlines, was supported by the U. S. Department of Energy, Office of Energy Research,. We acknowledge LS-CAT for time at APS Sector 32. Use of the National Magnetic Resonance Facility at Madison was supported in part by NIH grant RR02301 from the National Center for Research Resources (NCRR). This work is also based upon research conducted at the Northeastern Collaborative Access Team (NE-CAT) beamlines of the Advanced Photon Source, supported by NIH grant RR15301 from the NCRR. ‡ Atomic coordinates, along with structure factors for the X-ray structures and constraint lists for the NMR conformers, have been deposited in the Protein Data Bank, www.rcsb.org, under PDB # 2EVN (NMR structure of At1g77540), PDB # 1XMT (X-ray structure of At1g77540), and PDB # 2GDB (X-ray structure of At1g77540-CoA complex). Resonance assignments and primary NMR data have been deposited in BioMagResBank, www.bmrb.wisc.edu, under Arabidopsis thaliana At1g77540.1 for structural characterization on the basis of its target selection algorithm. This protein (At1g77540) consists of 103-residues (11.6 kDa) and had less than 25% sequence identity to any other structure deposited in the Protein Data Bank. In addition, no biochemical function had been established for this protein or its close homologs. However, the Superfamily server (1) predicted a very weak ...

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Auto-induction medium for the production of [U-15N]- and [U-13C, U-15N]-labeled proteins for NMR screening and structure determination

Cited by 93 publications

References 30 publications

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

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

Optimized expression and purification of myristoylated human neuronal calcium sensor 1 in E. coli

Structure of Arabidopsis thaliana At1g77540 Protein, a Minimal Acetyltransferase from the COG2388 Family^,

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Auto-induction medium for the production of [U-15N]- and [U-13C, U-15N]-labeled proteins for NMR screening and structure determination

Cited by 93 publications

References 30 publications

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

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

Optimized expression and purification of myristoylated human neuronal calcium sensor 1 in E. coli

Structure of Arabidopsis thaliana At1g77540 Protein, a Minimal Acetyltransferase from the COG2388 Family,

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Structure of Arabidopsis thaliana At1g77540 Protein, a Minimal Acetyltransferase from the COG2388 Family^,