Strategies for the Production of Recombinant Protein in Escherichia coli

Author:

Strategies for the Production of Recombinant Protein in Escherichia coli (1)

A protein’s intrinsic ability to fold, its solubility, preferential codon use, its toxicity, and its need for post translational modification have influence on expression of foreign gene in E. coli. At least four strategies can useful for increasing the expression and solubility of over-expressed protein; Changing the vector, changing the host strain, and adding of some chemicals during the induction or co-expression of other genes may help in the proper folding of the desired protein.

The following may work to overcome these problems, Gene sequence alteration include the removal of a signal peptide coding sequence, optimisation of codon according to E. coli, deletion of hydrophobic patch coding sequences, and prevention of stable mRNA secondary structure formation by changing the sequence which forms secondary structure etc.

Changing the vector refers to either changing the promoter with which the gene of interest is to be cloned or changing the fusion ‘tag’ which influence solubilization of recombinant protein in E. coli host. Consecutive 6-Histidine can possibly decrease the solubility of a fused protein. HAT fusion proteins, which is 19 amino acids long and has a non adjacent 6-Histidine, exhibit solubility close to wild-type proteins without compromising the affinity for metal. pET-39b and pET-40b plasmid are used to conjugate 208 aa DsbA and 236 aa DsbC proteins involved in periplasmic disulphide bond formation respectively. DsbA helps in the formation of disulphide

bond and DsbC assists in the isomerization of disulphide bond. The recombinant protein(s) conjugated with this protein tag can form disulphide bond in E. coli. pREP4 plasmid encodes lac repressor which ensures tight regulation of expression. Sometimes periplasmic translocation of gene products can be required for proper folding and/or disulphide bond formation or for easy purification. Non expression of gene is due to frame shift in the reading frame during cloning which causes stop codon incorporation.

Many biotech companies provide different types of genetically altered E. coli strain as per suitability of expression of foreign genes. The BL21 strain is the preferred choice for expression because of absence of two main proteases genes cloned in pET system with T7 promoter should be expressed in BL21(DE3). E. coli strain BL21(DE3) has a T7 polymerase encoding gene introduced in its genome as well as Lon and OmpT protease deleted. OrigamiTM E. coli strain of ‘Novagene’ may be used if disulphide bond is required for proper folding of protein. The Origami strain is an oxidising E. coli strain with mutation in thioredoxin reductase (trxB) and glutathione reductase (gor) genes that allows disulphide bond formation within cytoplasm. Lemo21(DE3) strain is used for the expression of difficult proteins such as membrane protein, toxic protein or the protein that aggregates in E. coli without compromising their stability and function. High level expression usually causes formation of an inclusion body. One way to avoid or decrease the inclusion body formation is to reduce cultivation temperature of the host after induction.

The solubility of recombinant protein is increased by prolonged induction at low temperatures with decreased amounts of IPTG. Expressed proteins may be stabilized by the binding with ligands (a small chemical agent) and have found widespread application in recombinant protein productions. Removal of signal peptide coding sequence also increases the expression and stability of recombinant protein. The deletion of a transmembrane or hydrophobic patch coding region increases expression of the gene and solubility of the recombinant protein.

Strategies for the expression of recombinant proteins in E. coli can be prioritized by changing the following factors (1) cultivation parameters, (2) host strain, (3) vector, (4) co-expression, (5) ORF with unaltered functional domain/structure. It would be beneficial to screen the solubility of recombinant protein through available in silico tools before cloning into particular vectors. ArcticExpress codon plus and Rossetta-gami would be ideal if one E. coli strain could possess all the characteristics required for heterologous gene expression such as, disulphide bond formation, rare t-RNA, low temperature adapted chaperone, slow expression etc.

1. G. J. Gopal, A. Kumar, Strategies for the production of recombinant protein in Escherichia coli. Protein J. 32, 419–425 (2013).

Leave a Reply

Your email address will not be published. Required fields are marked *