The N‐domain ofEscherichia coliphosphoglycerate kinase is a novel fusion partner to express aggregation‐prone heterologous proteins

Abstract

As a fusion partner to express aggregation-prone heterologous proteins, we investigated the efficacy of Escherichia coli phosphoglycerate kinase (ePGK) that consists of two functional domains (N- and C-domain) and reportedly has a high structural stability. When the full-length ePGK (F-ePGK) was used as a fusion partner, the solubility of the heterologous proteins increased, but some of them still had a large fraction of insoluble aggregates. Surprisingly, the fusion expression using the N-domain of ePGK (N-ePGK) made the insoluble fraction significantly reduce to less than 10% for all the heterologous fusion proteins tested. Also, we evaluated the efficacy of N-ePGK in making the target proteins be expressed with their own native function or structure. It was found that of human ferritin light chain, bacterial arginine deiminase, human granulocyte colony stimulating factor were synthesized evidently with the self-assembly function, L-arginine-degrading activity, and the correct secondary structure, respectively, through the fusion expression using N-ePGK. These results indicate that N-ePGK is a highly potent fusion partner that can be widely used for the synthesis of a variety of heterologous proteins in E. coli.

Production of prone‐to‐aggregate proteins

“…Other noteworthy fusion partners are the N-Domain of E. coli phosphoglycerate kinase, a 22 kDa protein domain, recently reported by the Lee group to enhance solubility of several proneto-aggregate proteins [28], and two additional tags: the modified bacteriophage T7 protein kinase and the E. coli Skp chaperone, analyzed by the Esposito group [29]. These tags were tested in combination with several prone-to-aggregate targets, and exhibited enhanced solubility of the target proteins even after cleavage of the fusion tags.…”

YrhB is a highly stable small protein with unique chaperone‐like activity in Escherichia coli BL21(DE3)

“…YrhB was used as N‐terminus cis ‐element of aggregation‐prone heterologous proteins that are expressed in E. coli BL21(DE3). Human epidermal growth factor (EGF), human prepro‐ghrelin (ppGRN), human interleukin‐2 (hIL‐2), human activation induced cytidine deaminase (AID), human glutamate decarboxylase (GAD 448–585 ), Mycoplasma arginine deiminase (ADI), human granulocyte colony‐stimulating factor (G‐CSF), human ferritin light chain (hFTN‐L), and cold autoinflammatory syndrome 1 protein Nacht domain (NACHT) have been reported to easily form inclusion bodies when directly expressed in E. coli [29]. We tried to synthesize the fusion protein, N ‐[YrhB]‐[heterologous protein]‐ C using YrhB as an N‐terminus cis ‐element.…”

High cell density cultivation of a recombinant Escherichia coli strain expressing a 6-O -sulfotransferase for the production of bioengineered heparin

“…The lack of appropriate folding‐assistant proteins and a post‐translational modification system in E. coli often causes aggregation and low productivity of recombinant eukaryotic proteins (Song et al . ). The engineered strain was used to express the catalytic domain of mouse 6‐OST‐3 (Pro121‐Pro450), a transmembrane protein.…”