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The Length of Polypeptide Linker Affects the Stability of Green Fluorescent Protein Fusion Proteins

1999; Elsevier BV; Volume: 273; Issue: 2 Linguagem: Inglês

10.1006/abio.1999.4235

1096-0309

Mark Prescott, Szczepan Nowakowski, Phillip Nagley, Rodney J. Devenish,

Viral Infectious Diseases and Gene Expression in Insects

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.