Fengchun Ye

Active inclusion bodies of acid phosphatase PhoC: aggregation induced by GFP fusion and activities modulated by linker flexibility

BackgroundBiologically active inclusion bodies (IBs) have gained much attention in recent years. Fusion with IB-inducing partner has been shown to be an efficient strategy for generating active IBs. To make full use of the advantages of active IBs, one of the key issues will be to improve the activity yield of IBs when expressed in cells, which would need more choices on IB-inducing fusion partners and approaches for engineering IBs. Green fluorescent protein (GFP) has been reported to aggregate when overexpressed, but GFP fusion has not been considered as an IB-inducing approach for these fusion proteins so far. In addition, the role of linker in fusion proteins has been shown to be important for protein characteristics, yet impact of linker on active IBs has never been reported.ResultsHere we report that by fusing GFP and acid phosphatase PhoC via a linker region, the resultant PhoC-GFPs were expressed largely as IBs. These IBs show high levels of specific fluorescence and specific PhoC activities (phosphatase and phosphotransferase), and can account for up to over 80% of the total PhoC activities in the cells. We further demonstrated that the aggregation of GFP moiety in the fusion protein plays an essential role in the formation of PhoC-GFP IBs. In addition, PhoC-GFP IBs with linkers of different flexibility were found to exhibit different levels of activities and ratios in the cells, suggesting that the linker region can be utilized to manipulate the characteristics of active IBs.ConclusionsOur results show that active IBs of PhoC can be generated by GFP fusion, demonstrating for the first time the potential of GFP fusion to induce active IB formation of another soluble protein. We also show that the linker sequence in PhoC-GFP fusion proteins plays an important role on the regulation of IB characteristics, providing an alternative and important approach for engineering of active IBs with the goal of obtaining high activity yield of IBs.

Rational design of a tripartite fusion protein of heparinase I enables one-step affinity purification and real-time activity detection

Enzymatic degradation of heparin has great potential as an ecological and specific way to produce low molecular weight heparin. However, the commercial use of heparinase I (HepA), one of the most important heparin lyases, has been hampered by low productivity and poor thermostability. Fusion with green fluorescent protein (GFP) or maltose-binding protein (MBP) has shown potential in facilitating the industrial use of HepA. Thus, tripartite fusion of GFP, MBP and HepA would be a promising approach. Therefore, in the present study, the tripartite fusion strategy was systematically studied, mainly focusing on the fusion order and the linker sequence, to obtain a fusion protein offering one-step purification and real-time detection of HepA activity by fluorescence as well as high HepA activity and thermostability. Our results show that fusion order is important for MBP binding affinity and HepA activity, while the linker sequences at domain junctions have significant effects on protein expression level, HepA activity and thermostability as well as GFP fluorescence. The best tripartite fusion was identified as MBP-(EAAAK)(3)-GFP-(GGGGS)(3)-HepA, which shows potential to facilitate the production of HepA and its application in industrial preparation of low molecular weight heparin.