Transport of recombinant proteins

The transport of recombinant proteins is performed by signal peptides that are N terminal which are involved in targeting the protein that is expressed to a transport complex that is present in membrane. In order to reduce the impurities of transport protein these protein synthesis is targeted in the periplasm of host.

The other advantages of targeting proteins are obtaining protein that will contain the N terminal that is correct, decreased destruction of protein as the impurities in proteins are reduced and proteins are released in a simple manner [43, 44].

The periplasmic sequences that are used for exporting recombinant proteins are obtained from ompA, malE, pelB and many more [15, 45-52]. A sec translocase apparatus is used in order to export the proteins into the inner membrane [53, 54].

Folding of recombinant proteins:

The oxidizing environment of periplasm differs it from cytoplasm. In case of E.coli, the proteins that contain disulfide bonds that are stable are present in the envelope of cell and the Dsb proteins catalyze them. The fusion of Dsb or Dsc systems results in the formation of disulfide bonds of proteins [25, 55, 56].

The unfolded proteins are supported from translocase apparatus and the folding modulators are Ppid, FpkA and PpiA and many more. The desirable folding activity is of FkpA along with the combination of enzyme PPIase.

The transfer of disulfide bond to the substrate of proteins is due to periplasmic protein that is DsbA. If the incorrect disulfide bonds are formed than DsbC protein is used in order to rescue and the isomerization is also catalyzed [63-64].

Secretion of recombinant proteins:

In case of most E.coli the pathways for the translocation of proteins are absent. While in some cases the periplasmic sequences move into the medium. Another pathway signal recognition pathway is used for the secretion of recombinant proteins [66, 67].

In case of bacteria the signal sequence of the proteins bind with the proteins that are excreted. The nascent chain complex of ribosomes than moves towards the membrane [68-70]. The ompA sequence is used for the secretion of recombinant proteins into the medium. In order to increase secretion the two factors of secretion like secE and secY are used [74-76].

Strategies for the release of recombinant proteins:

In order to produce recombinant proteins in E.coli the host should contain translational and transcriptional machinery. The load on expression machinery is increased when the recombinant proteins make 30% of the whole cell.

In order to increase the solubility of protein and its refolding the practices have been performed on regulation, function and structure of the aggregate of recombinant proteins in E.coli. These recombinant proteins can harm the host due to its overexpression and toxicity can be caused to host.

The capability of host cell to express proteins depends on metabolic load and it is affected by a number of factors [77, 78].

These factors are:

Expression of proteins when temperature is low:

The solubility of some complex proteins is increased when the temperature is low in case of E.coli. When the temperature is low than the stability of proteins is increased and also the pattern of folding is correct and these are due to hydrophobic interactions showing that the formation of inclusion bodies is dependent on temperature.

Another advantage is the toxic phenotypes that are expressed at 37C is found to be suppressed on lower temperature [79-81]. In E.coli the expression and activity of proteins is increased at low temperature and the expression of chaperons is also increased [82].

The proteases that are heat shock when induced during low temperature are poorly active. So, the temperature range between 15-23C prevents the degradation of proteins that are expressed [83, 84]. In E.coli the efficiency of promoters that are used in the expression of recombinant proteins is affected by lowering the temperature.

By using this technique the gene products that are unstable or the proteins that contain spanning domains can be carried successfully [85-87]. When we keep the temperature at 18C, the phosphodiestrase and at the temperature of 22C p38 is increased rather than keeping the temperature at 30-37C. Despite of the advantages of the low temperature the disadvantage of low temperatures are reduced rate of translation and transcription thus leading to reduced folding of recombinant proteins.