Selection of vector and host strain
Other important factors that may affect the solubility of a target protein is the selections of vector and host strain. Affinity tags are employed to improve protein solubility, prevent proteolysis and simplify the purification process. Maltose binding protein (MBP), N-utilizing substance A (NusA), prolyl cis-trans isomerases (PPIases), thioredoxin (Trx), intein, His-tag, glutathione-S-transferase (GST), and calmodulin-binding protein (CBP) are particularly suited for the soluble expression of proteins prone to form inclusion bodies. However, not all highly soluble proteins are suitable as solubility enhancers. Previous reports imply that E. coli MBP is a much more effective solubility partner compared to the highly soluble Trx or GST (Al-Hejin, Bora, & Ahmed, 2019; Sørensen & Mortensen, 2005). Additionally, in some cases, attaching polyionic peptide tags of the same charge to the protein of interest at a certain pH value could lead to increased protein solubility (Paraskevopoulou & Falcone, 2018). Several studies have shown that the nature of terminal residues in proteins can play a role in proteolytic degradation, denaturation and misfolding. Joining a C-terminal residue (17 aa) extension of Pfg27 to a target protein resulted in soluble expression and fold enhancement (Sørensen & Mortensen, 2005). The decreased solubility caused by consecutive (6×) histidine residues can be solved by using a pHAT vector with a lower overall charge and non-adjacent 6-Histidine. In our experiments, when we used pMAL system and pGEX vector, the maltose-binding protein (MBP) tag and GST tag fused to our target protein leading to overexpression and increased solubility of the protein. Since, the large size of a tag may interfere with the structure and function of the fused protein, multiple cleavage sites can be engineered flanking the expressed protein to remove the tag. Moreover, thioredoxin tag may enhance folding and disulfide bond formation of the target protein in strains lacking thioredoxin reductase (trxB) (Chang et al., 2014). Having been recently introduced by Choi et al., the pNew vector uses the cumate (4-isopropylbenzoic acid)-inducible expression system leading to a 3-6-fold increase in expression compared to the widely used pET expression system. Alternatively, the Wacker’s novel secretion technology results in the extracellular expression of soluble and properly folded proteins with high yield (up to 7.0 g/L) (Gupta & Shukla, 2016).
Numerous specialized host strains have been developed to express recombinant proteins in E. coli . For instance, the improved strains BL21(DE3)pLysS and BL21(DE3)pLysE both encode lysozyme in their genome as an inhibitor of T7 polymerases to prevent leaky expression. Similarly, CodonPlus-RIL and CodonPlus-RP strains provide a solution for the codon bias of AT- or GC-rich genes. On the other hand, Rosetta strain harbors all the genes encoding rare tRNAs eliminating the need for separate strains for the expression of AT- and GC-rich genes. Based on previous research, providing the rare tRNAs for the host cell promotes the expression level of soluble protein (Ni et al., 2019).
Oxidative environment is necessary for the formation of disulfide bonds. The Origami™(DE3) strain of E. coli developed by Novagen can be used to form disulfide bonds for correct folding of disulfide-bond dependent proteins. In addition to trxBand gor mutations, the novel ‘SHuffle’ strain developed by New England Biolabs (NEB) harbors a DsbC chaperon within the cytoplasm for the expression of disulfide-bond-forming proteins (Baeshen et al., 2015; Berkmen, 2012). Molecular chaperones or appropriate binding partners are other options to be considered. Lastly, E. coli mutant strains C41(DE3) and C43(DE3) are good choices for soluble expression of globular or membrane proteins (Rosano & Ceccarelli, 2014).