title: Active inclusion bodies of acid phosphatase PhoC: aggregation induced by GFP fusion and activities modulated by linker flexibility.
abstract: BACKGROUND
  Biologically 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.
  
  
  RESULTS
  Here 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.
  
  
  CONCLUSIONS
  Our 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.
authors.affiliation: Key Laboratory for Industrial Biocatalysis, Ministry of Education, Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China.
authors.forename: Ziliang
authors.initials: Z
authors.lastname: Huang
authors.forename: Chong
authors.initials: C
authors.lastname: Zhang
authors.forename: Shuo
authors.initials: S
authors.lastname: Chen
authors.forename: Fengchun
authors.initials: F
authors.lastname: Ye
authors.forename: Xin-Hui
authors.initials: XH
authors.lastname: Xing
mesh_terms: D000135:Acid Phosphatase / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D002479:Inclusion Bodies / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D010770:Phosphotransferases; D000135:Acid Phosphatase