Key result
A GFP fusion tag facilitated the soluble expression and correct folding of a pea actin isoform (PEAc1) in E. coli, improving its solubility and actin-associated activities compared to non-fused PEAc1.
GFP fusion promotes the soluble expression and correct folding of the pea actin isoform PEAc1 in E. coli.
GFP fusion boosts PEAc1 solubility in E. coli; leaves open utility for cardiovascular cytoskeletal protein studies.
In the present study, we report that a GFP fusion tag facilitated the soluble expression of a pea actin isoform (PEAc1) in E. coli. To investigate the influence of a GFP fusion tag on PEAc1 structure and activity, PEAc1, His-tagged PEAc1 (His-PEAc1), His-tagged GFP (His-GFP), and His-tagged PEAc1 fusion with GFP (His-PEAc1-GFP) were expressed in E. coli. SDS-PAGE and western blot analyses reveal that the solubility of His-PEAc1-GFP was higher than that of PEAc1 and His-PEAc1. The His-PEAc1-GFP and His-GFP fusion proteins were purified from the supernatant of cell homogenate on a Ni-affinity column, and PEAc1 and His-PEAc1 were purified from inclusion bodies. CD spectrum analysis of the four purified proteins indicated that the proportion of α-helix and β-sheet in PEAc1 was closest to the predicted data in His-PEAc1-GFP (compared with His-PEAc1 or PEAc1). In addition, the actin-associated activities of His-PEAc1-GFP, including polymerization to microfilaments under specific ionic conditions and DNase I inhibition by monomers, were more similar to those of muscle actin (compared with PEAc1 and His-PEAc1). These improvements in PEAc1 solubility and activity are likely the result of correct PEAc1 folding mediated by GFP fusion.
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Zhang et al. (2022) studied this question. GFP fusion tag vs. PEAc1 and His-PEAc1 was evaluated on Solubility and actin-associated activities. A GFP fusion tag facilitated the soluble expression and correct folding of a pea actin isoform (PEAc1) in E. coli, improving its solubility and actin-associated activities compared to non-fused PEAc1.
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