An Algorithm for Protein Refolding Technical Brief
Sheldon E. Broedel, Jr., Ph. D.
Athena Environmental Sciences, Inc., Baltimore , MD
With the extensive array of different approaches which have been reported for
the refolding of proteins, it can be a challenge to decide which approach to
pursue for any given protein. While in silico models may be helpful in
identifying possible solutions, they are not yet sufficiently refined to
predict the best conditions for refolding a protein. Therefore, an empirical
approach remains the most reliable path to defining the refolding conditions.
There are several parameters which affect the folding of proteins. Most
notable are the buffer composition, which includes: (1) Protein concentration,
type of buffer, pH, ionic strength, presence of excipitents, and the redox
potential, (2) physical parameters such as temperature and time, and (3) the
method for refolding, for example using dilution, dialysis, chromatographic,
and immobilization techniques either with a single- or double-step buffer
exchange. The number of possible combinations is consequently quite high
making it impossible to test all of the permutations. To assist in defining
the conditions for the refolding of a protein, we have devised an algorithm
for screening and then optimizing the method. The strategy is to quickly
identify the most likely means for refolding the target protein by first
identifying a working buffer system and then eliminating those approaches
which are not yielding refolded protein.
The basic scheme for the refolding algorithm is shown on the following page.
The scheme relies on a buffer screen which is designed to identify the
critical components for proper refolding. A graded set of tests are
performed beginning with a simple refolding-by-dilution method and
progresses to a more technically difficult refolding-by-chromatography
method. In the first round of testing, a set of 15 buffers is used in a
single-step refolding by dilution experiment. This is the quickest and
simplest method for identifying a suitable buffer and the critical components.
Since it is more amenable to a high throughput format, those buffers which do
not work can be ruled out early in the process. This makes subsequent
experiments with alternative refolding techniques more manageable. Using a
subset of buffers, the refolding is repeated by modulating the physical
parameters and then testing the potential utility of the different refolding
techniques, i.e., standard dialysis, slow dialysis (buffer exchange at 1
ml/min over 48 hours), and chromatographic (affinity, ion exchange or size
exclusion). Once the critical parameters for refolding have been identified,
the buffer and physical parameters are optimized using a range-finding
experimental design followed by a more narrow titration of the specific
reagents/conditions to determine the optimum refolding.
There are cases were a single-step refolding technique does not work.
In such situations, it is advised that a two-step refolding method be tested.
The two-step refolding approach typically uses a dilution technique.
In the first step the denatured protein is diluted into a buffer containing
a detergent. The choice of detergent is protein-specific and must be determined
empirically. After a period of time, one of several different types of
cyclodextrins is added to sequester the detergent. Again, the choice of
cyclodextrin is protein-specific. The critical buffer composition and
physical parameters can be identified using a high throughput screen format
with a dilution method. The process is then optimized, as described above,
for refolding by dilution, dialysis or chromatography depending on the needs
of the protein.
Figure 1. Flow chart of an algorithm to optimize protein refolding.
The initial step is to screen several buffer compositions to identify
the critical factors affecting the refolding of a target protein. A one or
two-step procedure may be needed depending on the protein. Once the basic
buffer composition is defined the physical parameters affecting refolding are
determined. To permit scaling of the refolding alternative techniques such as
slow dialysis, diafiltration, and immobilization are tested and the most suitable
selected. Finally, the system is optimized and scaled to the level needed.