Protein Production and Crystallization at SECSG – An Overview

Wang, Bi Cheng; Adams, Michael W. W.; Dailey, Harry A.; DeLucas, L.J.; Luo, Ming; Rose, John P.; Bunzel, R.; Dailey, Tamara A.; Habel, J.; Horanyi, P.S.; Jenney, Francis E.; Kataeva, Irina; Lee, H.; Li, Songlin; Li, Ting; Lin, Dazhen; Liu, Zhi Jie; Luan, Chi Hao; Mayer, Michael R.; Nagy, Lisa; Newton, M. Gary; Ng, Joseph D.; Poole, Farris L.; Shah, Ashit; Shah, C.; Sugar, F.J.; Xu, Hao

doi:10.1007/s10969-005-2462-z

Cited by 17 publications

(9 citation statements)

References 15 publications

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“…However, in general efforts have been concentrated on individual organisms. For an example, the Southeast Collaboratory for Structural Genomics has developed high throughput protein production and crystallization of genes originating from Pyrococcus furiosus [26]. More recently, a series of diatom expression vectors based on the Invitrogen Gateway technology for high throughput protein tagging and overexpression in Phaeodactylum tricornutum has been described [27].…”

Section: Discussionmentioning

confidence: 99%

MARINE-EXPRESS: taking advantage of high throughput cloning and expression strategies for the post-genomic analysis of marine organisms

et al. 2010

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BackgroundThe production of stable and soluble proteins is one of the most important steps prior to structural and functional studies of biological importance. We investigated the parallel production in a medium throughput strategy of genes coding for proteins from various marine organisms, using protocols that involved recombinatorial cloning, protein expression screening and batch purification. This strategy was applied in order to respond to the need for post-genomic validation of the recent success of a large number of marine genomic projects. Indeed, the upcoming challenge is to go beyond the bioinformatic data, since the bias introduced through the genomes of the so called model organisms leads to numerous proteins of unknown function in the still unexplored world of the oceanic organisms.ResultsWe present here the results of expression tests for 192 targets using a 96-well plate format. Genes were PCR amplified and cloned in parallel into expression vectors pFO4 and pGEX-4T-1, in order to express proteins N-terminally fused to a six-histidine-tag and to a GST-tag, respectively. Small-scale expression and purification permitted isolation of 84 soluble proteins and 34 insoluble proteins, which could also be used in refolding assays. Selected examples of proteins expressed and purified to a larger scale are presented.ConclusionsThe objective of this program was to get around the bottlenecks of soluble, active protein expression and crystallization for post-genomic validation of a number of proteins that come from various marine organisms. Multiplying the constructions, vectors and targets treated in parallel is important for the success of a medium throughput strategy and considerably increases the chances to get rapid access to pure and soluble protein samples, needed for the subsequent biochemical characterizations. Our set up of a medium throughput strategy applied to genes from marine organisms had a mean success rate of 44% soluble protein expression from marine bacteria, archaea as well as eukaryotic organisms. This success rate compares favorably with other protein screening projects, particularly for eukaryotic proteins. Several purified targets have already formed the base for experiments aimed at post-genomic validation.

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Section: Discussionmentioning

confidence: 99%

MARINE-EXPRESS: taking advantage of high throughput cloning and expression strategies for the post-genomic analysis of marine organisms

et al. 2010

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“…proposed LIE (linear interaction energy) algorithm to calculate absolute and relative Gibbs free energies from following equation:

Δ G_{bind} = α ((), V_{bound}^{normale normall} - V_{free}^{normale normall}) + β ((), V_{bound}^{normalv normald normalw} - V_{free}^{normalv normald normalw})

where “vdw” and “el” refer to the van der Waals and electrostatic energies between the drug and CNT in free (system without CNT) and bounded systems (system numbers 21–40 of Table ). Values of α = 0.5 and β = 0.16 were set for the energy calculation . γ is neglected in this equation to compare the relative binding energies.…”

Section: Theoretical Methodsmentioning

confidence: 99%

Correlation of Drug and Carbon Nanotube Size in Encapsulation and Free Energy Calculation: A Molecular Insight

Ghadamgahi

Ajloo

2015

Bulletin Korean Chem Soc

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Spontaneous encapsulation of drugs into carbon nanotubes (CNTs) has attracted great interest because of their importance in biological and biomedical devices. In this work, the diameter dependence of 20 drugs on the carbon nanotube size was explored via molecular dynamics (MD) simulation. Negative free energy of interaction, reduced potential of mean force, reduced CNT-drug distance, and reduced number of water molecules after encapsulation confirm the encapsulation of the drug into the smallest possible size of CNT. The variations of the radius of gyration of the CNTs and drugs were compared to explore the correlation trend. One of the factors influencing the encapsulation and insertion of drug is the size of the nanotube. In addition, the linear correlation between the drug and the nanotube size was confirmed by quantitative structure-property relationship (QSPR) analysis, and the multiple linear regression (MLR) method was applied to develop the correlation model. The regression parameter provided by the MLR method was R 2 = 0.99 for prediction of the drug gyration radius. The MLR prediction confirms that the larger drug molecule prefers to locate inside a larger CNT, which agrees with MD data. It was found that there is π-π interaction between the oxygen atoms and the aromatic ring of some of the drugs and the aromatic rings of CNTs are conjugated; this helps the drug molecule to locate inside the CNT. These theoretical methods provide a simple, detailed, and alternative method to obtain optimal tube size for encapsulation.

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“…The work was done in collaboration with the Southeast Collaboratory for Structural Genomics (SECSG), where a large fraction of 2200 open reading frames in Pyrococcus furiosus have been cloned and purified by high-throughput techniques for structure determination for X-ray crystallography or NMR [57]. Small amounts of samples (3 ml) from a test sample set were loaded on a 25-well sample holder and frozen in liquid nitrogen.…”

Section: Synchrotron Radiation Xas and Xrf Approachesmentioning

confidence: 99%

Metallomics and metalloproteomics

Shi

Chance

2008

Cell. Mol. Life Sci.

135

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Metallomics and metalloproteomics are emerging fields addressing the role, uptake, transport and storage of trace metals essential for protein functions. The methodologies utilized in metallomics and metalloproteomics to provide information on the identity, quantity and function of metalloproteins are discussed. The most widely used approach is through inductively coupled plasma mass spectrometry to identify the metal bound to a protein, and electrospray ionization mass spectrometry to elucidate the structure, dynamics and function of a metal-protein complex. Other approaches include X-ray absorption and X-ray fluorescence spectroscopies, and bioinformatics sequence analysis. X-ray absorption spectroscopy utilizing a synchrotron radiation source is a powerful tool to provide a direct analysis of metal bound to proteins and proteomic metal distribution in biological matrices. With the advent of genome sequencing, a large database of protein primary structures has been established, and specific tools to identify metalloproteins in the genome sequences have been developed.

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Protein Production and Crystallization at SECSG – An Overview

Cited by 17 publications

References 15 publications

MARINE-EXPRESS: taking advantage of high throughput cloning and expression strategies for the post-genomic analysis of marine organisms

MARINE-EXPRESS: taking advantage of high throughput cloning and expression strategies for the post-genomic analysis of marine organisms

Correlation of Drug and Carbon Nanotube Size in Encapsulation and Free Energy Calculation: A Molecular Insight

Metallomics and metalloproteomics

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