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.
Department of Chemical Engineering, Tsinghua University, Beijing 100084, PR China.
Ziliang
Z
Huang
Fengchun
F
Ye
Chong
C
Zhang
Shuo
S
Chen
Yin
Y
Chen
Jingjun
J
Wu
Masahiro
M
Togo
Xin-Hui
XH
Xing
D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019763:Heparin Lyase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006495:Heparin, Low-Molecular-Weight / Q000378:metabolism; D058619:Maltose-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D006495:Heparin, Low-Molecular-Weight; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019763:Heparin Lyase