Strategies for the Production of Recombinant Protein in Escherichia coli (1)
In E. coli, we face two main problems; (1) is difficult or little expression of a foreign gene and (2) is solubility of recombinant proteins for over expression. At least four strategies can be useful for increasing the expression and solubility of overexpressed 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.
Changing the Vector
Consecutive 6-Histidine can possibly decrease the solubility of a fused protein. When a histidine tagged gene does not express the protein or recombinant protein forms inclusion bodies, the gene of interest should be cloned with GST tag (25 kDa), or maltose binding protein (MBP) tag (44 kDa). Fusion of 109-aa long Trx.tag thioredoxin protein also increases the solubility of conjugated protein. However, fusion tags like NusA, Trx and SUMO need an extra affinity tag for purification. The main disadvantage of NusA, Trx, and SUMO is their requirement for an extra affinity tag for protein purification. Solubility of recombinant protein in E. coli can be determined or checked with freely available software by conjugating the sequence of the fusion tag with the desired protein sequence.
Changing the Host Strain
Genes cloned with ‘tac’ promoter (pMAL system, pGEX system) can be expressed in the cloning host itself but 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). Leaky expression of toxic genes is detrimental to the host cell—the host cell will not survive or it will change its mechanism so that even in presence of an inducer it will not allow production of toxic protein. The Origami strain is an oxidizing E. coli strain with mutation in thioredoxin reductase (trxB) and glutathione reductase (gor) genes that allows disulphide bond forma- tion within cytoplasm. 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.
Changing the Culture Parameters of Recombinant Host Strain
This concept is exploited to increase the proportion of recombinant protein expressed in soluble form or to stabilize a protein during purification. Ethanol, heat shock i.e. temperature treatment, benzyl alcohol, osmolytes, ionic strength of buffer etc., are inducers that enhance expression and production of recombinant proteins.
Changing Gene Sequences Without Changing the Functional Domain of Protein
Removal of signal peptide coding sequence also increases the expression and stability of recombinant protein. To avoid the formation of this secondary structure, codon sequences can be manipulated via site directed mutagenesis without changing the amino acid sequence. Commercial gene optimization services that optimize gene sequences and then synthesize that gene for efficient expression in a given host are also available. Usually gene optimization software consider following parameters: (a) Elimination of cryptic splice sites and RNA destabilizing sequence elements for increased RNA stability, (b) Addition of RNA stabilizing sequence elements, (c) Codon optimization and G/C content adaptation for your expression system, (dld) Intron removal and avoidance of stable RNA secondary structures.
When recombinant protein is expressed immensely in bacteria, it tends to misfold and accumulate as a soluble and insoluble nonfunctional aggregate. A general strategy to improve the native folding of recombinant proteins is to increase the cellular concentration of viscous organic compounds, osmolytes, or of molecular chaperones that can prevent aggregation. Strategies for the expression of recombi- nant 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