Improving the solubility of proteins

The strain of E.coli that is routinely used for the expression of proteins is BL21. The strain BL21 is deficient in OmpT and lon and this deficiency is a genetic modification which is used to increase the stability of proteins [88]. The derivative of BL21 is BLR which is involved in the stability of target plasmids having the sequences that are repetitive or the location where such products cause the loss of prophage [89-91].

The uniform expression of proteins in all cells is obtained by causing deletion mutation in BL21. A strain is created which is responsible for the entry of IPTG into cells [92], thus the levels of IPTG are controlled by using this modified strain. There are some new strains which contain gor gene and torx removed thus causing the greater disulfide bond formation.

In order to overcome the problems of over expression of tRNA another strain Rosetta is used. The solubility of proteins is increased by using strains of BL21 which have the deficiency of Ompt and lon genes [10, 93].

Obtaining soluble protein:

The level to which recombinant protein is accumulated in E.coli depends upon:

·         Specifity of activity.

·         Requirements for growth factor.

·         Final density of cell.

 

When the recombinant protein is produced in batch culture than there is the requirement of nutrients that are essential for growth from the very start and so, we can control growth parameters in limited manner.

In this process the ph is changed, amount of dissolved oxygen is changed, depletion of substrate is changed and inhibitory agents are changed. These changes are necessary if we want to produce recombinant protein and also the protein with correct folding. As on the nature of the recombinant protein that is expressed the efficient folding of protein requires: in growth media presence of specific co factors like iron-sulfur co factors and FMN.

When these co factors are added into the batch culture than the production of recombinant protein is increased and also the folding of protein is proper [97, 98]. In order to develop expression media that would prove best requires many permutations involving experimental. When we desire to perform experimentally than different techniques are available that can be used to prepare expression media that would be best.

In order to produce recombinant protein than research scheme is required for the production of medium that would be best in the experiment [99, 100]. The composition of medium is essential as it is directly related to the production of protein that is soluble. We have utilized SOC and LB to maximize the expression of soluble protein.

Different medias are modified to get better results and better activities. Because of these changes or modifications has decreased the time of expression, elevated the fraction of soluble protein and the activity of enzymes is also changed.