During the last period, the demand for enzymes and antibodies has increased, since they began to be widely used in medicine and biotechnology. But, the used of these proteins raises problems of structural stability, biochemical interactions and the shelf lives (2). In this context, the technology used for the stabilization and refolding of the tertiary structure of proteins have been become important.
In this review, we present the techniques for proteins stabilization and refolding that could improve the test’s quality of hepatitis C (HCV – hepatitis C virus). In the first instance, we will present the principles of test for hepatitis C (HCV ELISAs) (1), then, we will discuss the connections between the characteristics of the tertiary structure of proteins and their biochemical interactions. Finally, the methods of stabilizing and refolding proteins will be presented (1, 3, 4).
HCV ELISA identifies the presence of anti-HCV antibody. To analyze the stability of protein the positives tests are used. For immunological detection of anti-HCV antibodies in human sera, two differently modified forms of the HCV/NS3 (helicase) and CV/NS4 antigens (synthetic peptide) are used. The human sera are incubated together with the antigens to which a ruthenium complex was attached. The ruthenium complex allows electrochemiluminescence detection (1).
A frequent problem that hinders the routine application of these highly sensitive assays is the stability of the antigens. Due to several factors, antigens lose their tertiary structure (unfolding) and expose hydrophobic chains involved in non-specific interactions. To be used in ELISA test, they should yield reproducible signals even after prolonged storage and incubation. Another problem frequently encountered in these assays is that the proteins or peptides have a limited shelf life (2). So, the methods for stabilizing and refolding the proteins are needed.
There are several techniques for folding of proteins, each with advantages and drawbacks.
Direct dilution: the simple dilution in a specific buffer is a method simple for refolding proteins at the laboratory scale, but this technique has serious drawbacks during scale-up as huge refolding vessels and additional cost-intensive concentration steps are required after renaturation (3).
Membrane controlled denaturant removal: resemble with direct dilution method, but the change from denaturing to native buffer conditions occurs gradually. More aggregation during refolding compared to the direct dilution method and the refolding is negatively affected by non-specific adsorption are two this method’s disadvantages (3).
Protein refolding based on size exclusion chromatography: it is an exclusion chromatography for a denaturant-protein solution. A refolding buffer is used in the first step (equilibration) and the second step (elution). The method is more efficient than the simple dilution, because at the same time, refolding and the concentration of the proteins are made (3).
Matrix-assisted protein refolding. The solubilised and unfolded protein are attached to a solid support, then matrix-protein complex is brought to refolding conditions such as dilution, dialyse or chromatography and finally, the refolded protein are detached from the matrix. Due to the selective binding, matrix-assisted refolding can combine the renaturation of the target protein with its purification from the proteins contaminants (3).
Physical aiding protein refolding: in general, low temperatures support the productive folding pathway and hydrophobic aggregation is suppressed. During the depressurization step, proteins can reach their native state (3).
Chemicals protein refolding: L-arginine, detergents, glycerol, zwitterionic agents, micelles and liposomes are the chemicals used for stabilising and refolding the proteins. These substances suppress aggregation in favour of the productive folding pathway (3).
Biological protein refolding with the agents mimicking in vivo folding conditions: the chaperones are divided in two groups: ATP-independent Small Heat Shock Protein (sHsps) and two machineries ATP dependent (DnaK–DnaJ–GrpE and GroEL–GroES) (4). Their application is limited by their cost, the high concentration required and the need for their removal after the refolding procedure. sHsps can be used to stabilize proteins and the process is not expensive because it has a passive action (without ATP) (1, 4).
In conclusion, to stabilize the antigens and to increase the reproducibility of the successive tests, we must used the methods for stabilizing and refolding the proteins. We propose to use the sHsps and the low temperature for stabilising the antigens during the storage. If the quality of antigens decreased despite the presence of sHsps, we can restore the structure tertiary by method matrix-assisted protein refolding and finally adding the missing quantity of antigens. To analyze the quantity and the stability of protein the positives tests are used.
More from: Danny
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