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).