NMR paper Quantitation of protein expression in a cell-free system: Efficient detection of yiel

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[NMR paper] Quantitation of protein expression in a cell-free system: Efficient detection of yiel

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NMR processing:

MDD

NMR assignment:

Backbone:

Side-chains:

NOEs:

Structure from NMR restraints:

Ab initio:

Fragment-based:

Rosetta-NMR (Robetta)

Template-based:

GeNMR

I-TASSER

Refinement:

Amber

Structure from chemical shifts:

Fragment-based:

Homology-based:

Torsion angles from chemical shifts:

Preditor

TALOS

Promega- Proline

Secondary structure from chemical shifts:

CSI (via RCI server)

TALOS

MICS caps, β-turns

d2D

PECAN

Flexibility from chemical shifts:

RCI

Interactions from chemical shifts:

HADDOCK

Chemical shifts re-referencing:

NMR model quality:

NOEs, other restraints:

Chemical shifts:

RDCs:

Pseudocontact shifts:

Protein geomtery:

SAVES2 or SAVES4

Quality Control Check

NMR spectrum prediction:

FANDAS

MestReS

V-NMR

Flexibility from structure:

Backbone S2

Methyl S2

B-factor

Molecular dynamics:

Gromacs

Amber

Antechamber

Chemical shifts prediction:

From structure:

Shiftx2

Sparta+

Camshift

CH3shift- Methyl

ArShift- Aromatic

ShiftS

Proshift

PPM

CheShift-2- Cα

From sequence:

Shifty

Camcoil

Poulsen_rc_CS

Disordered proteins:

MAXOCC

Format conversion & validation:

CCPN

From NMR-STAR 3.1

Validate NMR-STAR 3.1

NMR sample preparation:

Protein disorder:

DisMeta

Protein solubility:

Isotope labeling:

Solid-state NMR:

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11-24-2010, 11:14 PM

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Quantitation of protein expression in a cell-free system: Efficient detection of yiel

Quantitation of protein expression in a cell-free system: Efficient detection of yields and 19F NMR to identify folded protein.

Related Articles Quantitation of protein expression in a cell-free system: Efficient detection of yields and 19F NMR to identify folded protein.

J Biomol NMR. 2005 Jan;31(1):11-9

Authors: Neerathilingam M, Greene LH, Colebrooke SA, Campbell ID, Staunton D

We have developed an efficient and novel filter assay method, involving radioactive labelling and imaging, to quantify the expression of soluble proteins from a cell-free translation system. Here this method is combined with the conformational sensitivity of 19F NMR to monitor the folded state of the expressed protein. This report describes the optimisation of 6-fluorotryptophan incorporation in a His-tagged human serum retinol-binding protein (RBP), a disulphide bonded beta-barrel protein. Appropriate reagent concentrations for producing fluorine labelled RBP in a cell-free translation system are described. It is shown that 19F NMR is a suitable method for monitoring the production of correctly folded protein from a high-throughput expression system.

PMID: 15692735 [PubMed - indexed for MEDLINE]

Source: PubMed

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