Microsoft powerpoint - protein expression and refolding.ppt
EXPLAINING PROTEIN EXPRESSION AND REFOLDING:
A step-by-step guide
This guide is intended to explain the basic steps and procedures involved in the expression,
refolding and purification of recombinant proteins. For more detailed information about designing
protein refolding protocols and the selection of suitable buffers components and conditions, you
should see the following resources on the Refold website:
• A practical guide to protein expression and refolding from inclusion bodies, Cabrita et al.
• Standard protocols
The processes involved in the expression and refolding of recombinant proteins can be described in a 5-step procedure, as outlined below:
– cloning and transformation into host cells
Protein expression – production of protein (using E.coli
Inclusion body preparation - cell lysis, inclusion body isolation and solubilization
NOTES: Protein expression and inclusion body preparation
SUMMARY: Generalised flowchart of protein production, refolding and purification
A. PRE-EXPRESSION PROCEDURES
Cloning and transformation into host cells
1. Replication of the target DNA gene.
2. Insertion of the target DNA gene into an
3. Transformation of the vector into the
The isolated target gene is then ligated into
protein is specifically reproduced by a suitable plasmid vector. The vector is
used is E.coli.
For each host organism,
Polymerase Chain Reaction (PCR) prepared by digestion using suitable
using complementary oligonucleotide restriction enzymes to produce ends
B. PROTEIN EXPRESSION
Production of protein (using E.coli)
1. Cells containing the expression vector are
3. The bacterial cell density is monitored by
added to media supplemented with nutrients
checking the spectroscopic absorbance of the
overnight, then a small amount (eg.10ml) is
absorbance at 600nm (A600/OD600) reaches a
added to a larger amount of media (eg.1L).
given value (usually 0.4-0.8) the cells have reached optimum growth density and, protein expression is induced. NB: sometimes different wavelengths are used, but generally close to 600nm.
4. Induction of Expression. Protein expression is
5. Following induction, the cell culture
usually initiated by the addition of an inducing
agent. The most common agent is isopropyl-D-
agents/methods include arabinose, nalidixic acid,
or increasing the incubation temperature.
C. INCLUSION BODY PREPARATION
Cell lysis, inclusion body isolation and solubilization
1. Pre-cell lysis incubation.
Cells may be frozen and thawed,
2. Cell lysis.
Cells are then broken open, or lysed. There are
then incubated at 4°C in the presence of lytic agents such as
several possible cell disruption methods. Examples of such
. Other lytic agents may include
methods include sonication, high pressure homogenization, French
commercial reagents such as BugBuster (Novagen)
press treatment, osmotic pressure
and freeze/thaw cycles
DNAseI may also be added to the cells to degrade DNA. Sometimes the cells are lysed directly without incubation
Cell lysis by
The lysed cells are centrifuged and the
The pelleted IBs are resuspended and
supernatant discarded. The remaining insoluble fraction, or
denatured in solubilization buffer, which contains high
pellet, contains the aggregated protein in inclusion bodies (IBs).
concentrations of denaturant (4-6M guanidine or 8M urea).
The IBs may be resuspended in a wash buffer and then
Alternatively, sometimes detergents (eg. N-lauroylsarcosine,
centrifuged again. The wash buffer may contain low
SDS) or changes in pH may be used to effectively unfold IBs.
concentrations of detergents (eg.Triton-X100, SDS) and/or
Generally the final volume of solubilization buffer is smaller than
denaturants (eg. guanidine, urea) This process may be repeated
the original resuspension volume of the cells. Sometimes the
mixture may be incubated with mixing for up to 2 hours or overnight to fully dissolve IBs. The solubilized IBs are centrifuged again and the supernatant is retained. The protein is now ready
NOTES: Protein Expression and Inclusion Body Preparation
Alternative cell lysis and solubilization – direct denaturation
Sometimes following expression and centrifugation, the whole cells can be resuspended directly in denaturant-containing solubilization buffer. The denaturant breaks down the bacterial cell wall, thus
effecting cell lysis, whilst simultaneously unfolding and solubilizing all of the proteins.
Sometimes proteins may be expressed as fusion proteins with a specific tag to facilitate purification or protein activation. Often the tag may be removed after purification or refolding by cleavage using a specific protease which recognises a specific unique sequence in the linker region.
Other examples of fusion tags:
N-terminal GST tag (facilitates affinity
purification) with a Factor Xacleavage site
Sometimes it is beneficial to further purify the solubilized inclusion body protein under denaturing conditions prior to refolding. The protein may be passed down a chromatographic column in its denatured/unfolded form, often in solubilization buffer or buffer containing equivalent quantity of denaturant. Such methods may include standard chromatographic processes (eg. Metal affinity, ion exchange, gel filtration, HPLC) The equilibration, wash and elution buffers used for the column contain high denaturant concentrations so the protein remains unfolded throughout the purification procedure.
D. PROTEIN REFOLDING
Following isolation and solubilization of the inclusion bodies, and maybe also pre-refolding purification, the protein is ready to be refolded. The basic principle of protein refolding is the removal of denaturant from the system. Proteins are refolded by an exchange of buffers, from denaturant-containing buffer (solubilization buffer) to no denaturant (refolding buffer). Refolding can be achieved in many different ways. The most methods are dilution, dialysis, metal-affinity chromatography (nickel-chelating, cobalt-chelating) and gel filtration/size exclusion chromatography. These common methods are described below.
Metal-chelating Column Refolding
This is a simple method in which enables the protein to
In this method the protein, usually expressed with a poly-
refold by diluting out the denaturant which is present.
histidine tag, is bound to a metal chelating column charged
Generally the denatured, solubilized protein is directly
with the appropriate metal ion (usually Ni2+ or Co2+). The
added to 20-100x fold volume of refolding buffer, and then
protein is loaded on and bound to the resin in its denatured
may be left for 4-16+ hours with stirring. A variation on the
unfolded state in solubilization buffer. The column is
basic dilution method is for the solubilized protein to be
generally washed to remove contaminants, and then the
added slowly to the refolding buffer (drip-drip) over a long
protein is refolded by the gradual removal of denaturant by
buffer exchange. This can be achieved by a linear gradient of decreasing denaturant, or alternatively, via a step-wise
gradient with a series of buffers each containing successively lower concentrations of denaturant. When the
The protein is dialysed against 40-100x fold volume of
buffer exchange and refolding is complete, the renatured
refolding buffer and is generally left for at least 8 hours. The
protein is then eluted from the column.
protein refolds by diffusion of denaturant molecules out of the dialysis tubing into the surrounding buffer. This results in
Size Exclusion/Gel Filtration Column refolding
dilution of the denaturant, and therefore the protein refolds. A variation on this simple is step-wise dialysis, whereby the
The unfolded protein (in solubilization buffer) is refolded by
protein is dialysed against a series of successive buffers,
application to a gel filtration column which is equilibrated
each containing a slightly lower concentration of denaturant
and run with refolding buffer. As the protein passes through
than the previous one, until the last buffer contains no
the column, the buffer is exchanged, the denaturant is
denaturant at all. Eg. 8 Æ 4 Æ 2 Æ 0M urea
E. POST-REFOLDING PROCEDURES
After the protein has been refolded, it is often further purified by chromatography. This may include affinity chromatography, ion-exchange chromatography, gel filtration chromatography, hydrophobic chromatography, HPLC
Fusion Tag Removal
If the refolded protein is a fusion protein, it may be incubated with a
specific protease to remove the fusion tag. This may also require another chromatographic step to separate the target protein from the
Sometimes the fusion tag is not cleaved, but remains attached to the
protein. This is more common with a hexa-histidine tag, as a relatively small number of amino acids have been added to the overall protein.
Pro-sequences are often removed after refolding so that the protein becomes fully active and functional.
Analysis of Refolded Protein
After refolding and purification, proteins are usually analysed to confirm the integrity of the product. ie. Is it pure? Is it in its native state, is it fully functional and active? Several different assays can be used to analyse the protein, depending on the protein. Such techniques may include:
Gel filtration/Size exclusion chromatography Circular
8-Anilo-1-Napthalenesulfonic Acid (Bis-ANS) fluorescence
High Performance Liquid Chromatography (HPLC)
SUMMARY: Generalised flowchart of protein
production, refolding and purification
Inclusion body isolation and solubilisation
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