Molecular chaperones

  

Molecular chaperones

The molecular chaperons are defined as the proteins that are adapted to increase folding of protein, increases conformation attainment and localization that is cellular and it does not becomes the portion of the final structure. The strategy of the co expression these molecular chaperones is used to prevent the formation of inclusion body and this causes the increased solubility of the expressed protein.

The chaperons that can be used are trigger factor that is involved in the folding of recombinant protein and another thing is that these polypeptides attain the folding that resembles the native state although they have been released from chaperon [103, 104]. Many other different chaperones are also used for this purpose [105-109]. We can also divide molecular chaperons into three classes:

Folding chaperones: These chaperones are involved in the refolding or un folding of the substrate through the change of shape by the help of ATP, in this way we can prevent the formation of inclusion bodies that leads to the increased formation of recombinant proteins and the destruction of those proteins that are misfolded [103, 104, 108, 109].

·         Holding chaperones: These chaperons work with folding chaperones to hold those proteins that are partially folded and they also prevent those proteins that are denatured by heat [110, 111].

·         Disaggregate chaperones: They are involved in the enhancement of solubility of aggregate of proteins [112-114].

Different chaperones are co expressed to obtain proteins in soluble form [103]. Some examples of chaperones are GroEL-GroES and many more and the co expression of these systems used along with trigger factor can increase the solubility of protein [115, 166].

The major requirements that are considered when we want to express eukaryotic proteins in any bacteria are:

·         The recombinant proteins may have effect on the metabolic system of the host and by doing this they can cause toxicity.

·         If they are expressing at the temperature of 37C than this temperature can cause the increased production of proteins that are obtained in insoluble form.

·         If the optimal media is lacking than majority of the recombinant protein is produced in insoluble form.

There are different factors which determine that protein will be produced in soluble form or insoluble form and some of these factors are cell mass, requirement of growth factors and the activity of protein that is expressed in bacteria.

Production of proteins by refolding of inclusion bodies:

Inclusion bodies are defined as they are the aggregates of proteins and these are formed when desired gene is over expressed in host organism like E.coli. When this aggregation of proteins is formed than it can cause un desirable folding of protein. These inclusion bodies are formed in the expression system of recombinant proteins when there is equilibrium between aggregation of protein and solubility. The accumulation of inclusion bodies is in either periplasm or cytoplasm [113, 116, 117].

Refolding of inclusion bodies:

If the recombinant proteins are expressed as inclusion bodies than we can easily purify them from E.coli. We can easily purify them through centrifugation because of their higher level of expression and their compactness. Few purification steps are required when we separate these proteins from E.coli because they are resistant to proteases which results in lower impurities and lower degradation and our efforts to obtain final product are reduced.

Although inclusion bodies are involved in the easy purification of proteins, the major disadvantage of this process is the loss of activity of proteins. Recombinant proteins expressed as inclusion bodies are widely used for the production of therapeutic proteins as the loss in activity which has occurred can be compensated by the higher level of expression [118-120].

The major challenge in this aspect is the conversion of insoluble and inactive proteins into more soluble form and also with correct folding.

The major challenges faced when we convert these inclusion bodies into active form are:

·         Reduced rate of discovery.

·         Requirement of certain conditions for the refolding of each protein.

·         The process of resolubilization can have negative impact on the activity of the protein.

The purification of soluble proteins by using inclusion bodies is time saving and easy process. The production of recombinant proteins is preferred over refolding of proteins.

Isolation of inclusion bodies:

We can obtain greater concentration of purified recombinant proteins when we isolate inclusion bodies. For this purpose lysozymes are used along with EDTA before we can carry out homogenization of cells in order to carry out disruption of cell. In order to recover inclusion bodies we use low speed centrifugation of the cells of bacteria and for small pieces, ultrasonication is carried out and for industrial scale, high speed homogenization is used.

Major fraction of inclusion bodies is obtained when cell envelop and outer membrane are precipitated in insoluble form [102, 121, 122]. In order to remove contamination we can add detergents like Triton-X100 and chaotropic compounds are added in low concentration and the mechanical disruption of the cells of bacteria is performed [123-127].

When we have removed impurities than inclusion bodies are solubilized by using various concentrations of chaotropic compounds like urea and many other agents like arginine. The compound that is normally preferred is guanidium hydrochlorides which is expensive but have better properties [128-133]. If we are using urea than its activity is dependent on ph and for each protein we have to determine optimum ph and this becomes a difficult task. We can also use solutions of urea which than produce cyanate and this can negatively affect the biological activity of protein [134-136].

A secondary structure is formed by proteins of inclusion bodies when they are present under mild conditions and their biological activity is retained up to a limit [137-140].It has been estimated that the solubility of protein under mild conditions lead to the best folding as compared to the solubility at high concentrations [141].

It has also been suggested that when ph values are extreme either the denaturants are present or absent [119, 142], causes the solubility of inclusion bodies. When the ph treatments are extreme than it can cause modifications like alkaline desulfuration and deamidation and it also changes the activity of the protein [143-145] and mostly scientists don’t choose this method.

In addition we can also use many other different solubilization agents like DTT and many more and they reduce the formation of unnecessary formation of disulfide bonds [136, 146-148]. If the proteins have disulfide bonds in their native forms than redox conditions are used.

If the alkaline ph is mild than there are optimum conditions for the disruption of already present disulfide bonds. Before refolding process the substances that reduce the residues and also have negative impact on the refolding of proteins are firstly removed. The reducing agents that are immobilized are also used and they are also involved in the removal of reducing agents. When the solubilization process is completed than this can be achieved by centrifugation. Before the removal of reducing agents the ph of the system is reduced in order to remove unnecessary formation of disulfide bonds.

Refolding of un-folded and solubilized proteins:

The methods which we use for the solubilization of inclusion bodies can cause the un desirable conformation of the proteins that are expressed. If we want to achieve native conformation than the proteins should be properly refolded. In the refolding of proteins the intra molecular interactions are involved and also first order kinetics.

The aggregation of proteins involves the interactions between the intermediates of protein folding and this is preferred at high concentrations of proteins. If the concentration of un folded protein is high than refolding of protein is decreased [149-151].

The refolding process does not involve these intermediates. It is desired that the initial concentration of un folded proteins should be low so that folding of the proteins can be favored. During the initial few steps of refolding the interactions are avoided and renatuartion is achieved when the protein concentration is high.