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Protocolo Active proteins

Preparation of active proteins from inclusion bodies.


Background:

It is often difficult to obtain soluble and active proteins from expression in prokaryotes. Often, overexpression leads to the production of inclusion bodies :insoluble aggregates of misfolded protein. These inclusion bodies can easily be purified. However the solubilization of the expressed protein can usually only be obtained using strongly denaturing conditions and a major problem is then to achieve an efficient folding in-vitro.

The problem is mainly how to lower the denaturant concentration to allow folding while at the same time preventing aggregation.

Most published methods suggest to eliminate the denaturing agent using dialysis. This is usually a mistake as dialysis exposes the protein solution to a decreasing denaturant gradient over a few hours. So proteins will remain exposed for an extended period of time to an intermediate denaturant concentration (2 to 4M Urea or guanidine) where they are not yet folded but no longer denatured and thus extremely prone to aggregation. This was shown as early as 1974 by a set of experiments by Prof. Goldberg and his team. In this paper (London et al. Eur.J.Biochem; vol. 37, pp409-415.), it was shown that the folding yield of tryptophanase would reach a minimum when the folding mix were incubated at intermediate denaturant concentration (see fig. 2 of this paper).

Next, it is important to limit the aggregation using mild solubilizing agents during the refolding steps. Non detergent sulfobetaines (NDSB) are well suited to this purpose and have been shown to be particularly efficient (Vuillard et al Biochem.J, (1995)., vol. 305 pp 337-343//Golberg et al. Folding and design (1996), vol. 1 pp21-27, s

The following protocol can be used as a starting point


Folding of proteins expressed as inclusion bodies.

1 Solubilization of proteins from inclusion bodies.

- The pellet from a litre of bacterial suspension is resuspended in 20ml of:

50mM HEPES-NaOH pH7.5, 0.5M NaCl, 1mM PMSF, 5mM DTT containing 0.35mg/ml lysozyme then incubated for 30 min at 20C.

- Triton X-100 is added to a concentration of 1% (vol./vol), this is followed by ultrasound sonication by bursts of 30sec followed by cooling until the solution clears.

- The extract is treated by Dnase I for 1h at 37C, enzyme concentration 20mg/l.

- The inclusion bodies are sedimented by centrifugation at 30 000g for 30min at 4C.

- The pellet (inclusion bodies) is washed twice with TBS or PBS containing 1% triton X 100 followed by spinning at 30 000g for 30min at 4C.

- The pellet (inclusion bodies) is the solubilized in 2 ml 50mM HEPES-NaOH pH7.5, 6M guanidine HCl, 25mM DTT and left for 1h at 4C.

- Insoluble material is removed by centrifugation at 100 000g for 10 minutes. This is important to remove existing aggregates that can act as nuclei to trigger aggregation during folding.

- Determine the protein concentration and adjust to 1mg/ml using 50mM HEPES-NaOH pH7.5, 6M guanidine HCl, 25mM DTT and proceed directly to folding.

2 Folding protocol.

The solubilized proteins are diluted as quickly as possible 1 in 10 into cold (4C) folding buffer:

50mM HEPES pH7.5, 0.2M NaCl, 1mM DTT, 1M NDSB256 (or 1M NDSB201).

the final protein concentration should not exceed 0.05 to 0.1 mg/ml. A fast an efficient mix is essential. for small volumes dispensing the protein solution with a pipette directly into the folding buffer while vortexing is adequate. (keep vortexing for 30 sec after addition) For larger volumes one can dispense the protein solution into the folding buffer using a syringe under vigorous ( magnetic stirrer) agitation. Keep stirring for 2 minutes after addition. Leave for 1h at 4C. The remaining guanidine and NDSB can then be removed by dialysis into any appropriate buffer.

3 Important notes.

- The key parameters are protein concentration, guanidine residual concentration and temperature. The values suggested here are only starting points, to optimise folding yields they may have to be optimised.

- Measuring the solution turbidity is a good indication of protein aggregation and, to compare conditions, it may be worth to measure the sample's absorbance at 400nm.

- NDSB256 Diemethylbenzylammonium propane sulfonate is usually slightly more efficient than NDSB201 [3(1-pyridinio)-1-propane sulfonate] but nDSB201 is much cheaper as it can be obtained from several suppliers.