Overcoming the Solubility Problem in E. coli: Available Approaches for Recombinant Protein Production

Protein engineering of a nanoCLAMP antibody mimetic scaffold as a platform for producing bioprocess-compatible affinity capture ligands

“…Our starting clone was an anti-SMT3 nanoCLAMP, SMT3-A1 ( 6 ), which we previously used as an affinity capture ligand for the laboratory-scale purification of SUMO-fusion proteins in a single step from whole cell extracts. The SUMO-fusion tag is widely used to improve the solubility and yield of proteins produced in E. coli and can be cleaved to leave behind a native sequence without any foreign amino acids ( 11 , 12 ). SMT3-A1 consists of residues 807 to 946 of the second Type 32 carbohydrate binding module of NagH from C. perfringens , with variable loops selected for binding to yeast SUMO.…”

Insights into the Alcyoneusvirus Adsorption Complex

“…As seen in Table 1, most of the recombinant RaK2 TFPs were obtained using E. coli Rosetta (DE3) cells, and only 10xHis_gp532 was produced in HMS174. Moreover, prolonged induction at lower temperatures was needed in most cases, except for 10xHis_gp533, which was induced at 30 • C. Although many recommendations on improving recombinant protein production and minimizing inclusion body formation may be found in the literature [65][66][67][68], the expression of individual TFPs, notwithstanding exhaustive efforts to optimize the expression conditions, is often unsuccessful [23,64,69,70]. In such cases, truncated protein variants are usually generated [23,[71][72][73].…”

“…It is important to note that all three recombinant proteins showed a propensity to aggregate after urea removal by dialysis. Although many recommendations on improving recombinant protein production and minimizing inclusion body formation may be found in the literature [65][66][67][68], the expression of individual TFPs, notwithstanding exhaustive efforts to optimize the expression conditions, is often unsuccessful [23,64,69,70]. In such cases, truncated protein variants are usually generated [23,[71][72][73].…”

Heterologous Expression of Difficult to Produce Proteins in Bacterial Systems

“…In the review published in this Special Issue, Kato summarizes the principles of this dual control and lists examples of this approach, including the use of site-specific unnatural amino acid incorporation, riboswitches, ribozymes, and antisense RNA [17]. Additionally, fusion tags have been widely used to reduce protein toxicity, also increasing protein solubility [18,19]. Another approach that can be used for these difficult-to-produce or toxic proteins is the cell-free or in vitro recombinant protein synthesis system, which has been extensively described by Smolskaya et al [20].…”