A novel multi-affinity tag system to produce high levels of soluble and biotinylated proteins in Escherichia coli

Oxyradical-induced GFP damage and loss of fluorescence

“…For PCR amplification of GFP, the primers were designed such that the resultant PCR product had an NdeI restriction site at the 5 end, and a BamHI site at the 3 end. This PCR product was digested with NdeI and BamHI after which it was ligated into pET-N-AT [34] vector which had been digested with NdeI and BamHI as well. DNA sequencing was used to confirm the sequence of resultant GFPconstruct, which had an N-terminal His 6 tag.…”

“…The his 6 -tagged GFP was purified as described previously [34]. Briefly, bacterial pellet was lysed by two cycles of freeze-thaw in 50 mM Tris, pH 8.0, and containing 0.1 mg/ml lysozyme.…”

EspA is a novel fusion partner for expression of foreign proteins in Escherichia coli

“…In addition, EspA is a highly immunogenic bacterial protein (Karpman et al, 2002), so it is necessary to remove the fusion partner, especially when producing fusion proteins for antibodies preparation. Therefore, a specific protease recognition site must be introduced into the EspA-fusion expression vector, although the removal of fusion tag presents an enormous challenge (Ashraf et al, 2004;Cao et al, 2003). Enterokinase, which recognizes the sequence DDDDK/X, was chosen due to its high specificity.…”

Polycationic amino acid tags enhance soluble expression of Candida antarctica lipase B in recombinant Escherichia coli

“…Even though some fusion partners provide the solubility enhancement of CalB in E. coli, some problems should be overcome such as proteolytic removal of big fusion partners and additional purification steps [9]. Because soluble fusion partners often have an inherent tendency to perturb both the structure and function of the target proteins due to their big size [10], the cleavage of soluble fusion partners frequently results in protein aggregation and yield loss [11].…”