title: A study on the effects of linker flexibility on acid phosphatase PhoC-GFP fusion protein using a novel linker library.
abstract: Fusion strategy has been widely used to construct artificial multifunction proteins. The flexibility or rigidity of linkers between two fused partners is an important parameter that affects the function of fusion proteins. By combining the flexible unit GGGGS (F) and rigid unit EAAAK (R), ten linkers consisting of five elementary units that cover the fully rigid RRRRR linker to the fully flexible FFFFF linker were used to construct acid phosphatase-green fluorescence protein fusion protein (PhoC-GFP). By varying the linker flexibility in PhoC-GFPs, the relative specific activity of phosphotransferase and phosphatase varied from ∼19.0% to 100% and ∼9.35% to 100%, respectively. There exists an optimal linker capable of achieving the highest phosphotransferase/phosphatase activity and GFP fluorescence intensity. We found that the highest activities were achieved neither with the rigid RRRRR linker nor with the flexible FFFFF linker, but with the FFFRR linker. Linker flexibility could adjust the activity ratio between phosphotransferase and phosphatase and varied between ∼30% to 100%. PhoC-GFP with FRRRR linker achieved the highest relative specific phosphotransferase activity/relative specific phosphatase activity (T/P) value. Our results show that applying a linker library with controllable flexibility to the fusion proteins will be an efficient way to adjust the function of fusion enzymes.
authors.affiliation: Key Laboratory of Industrial Biocatalysis, Ministry of Education, China; Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China.
authors.forename: Ziliang
authors.initials: Z
authors.lastname: Huang
authors.affiliation: Key Laboratory of Industrial Biocatalysis, Ministry of Education, China; Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China.
authors.forename: Gang
authors.initials: G
authors.lastname: Li
authors.affiliation: Key Laboratory of Industrial Biocatalysis, Ministry of Education, China; Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China. Electronic address: chongzhang@tsinghua.edu.cn.
authors.forename: Chong
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Industrial Biocatalysis, Ministry of Education, China; Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China.
authors.forename: Xin-Hui
authors.initials: XH
authors.lastname: Xing
mesh_terms: D000135:Acid Phosphatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000135:Acid Phosphatase
keywords: Acid phosphatase-green fluorescent protein; Enzyme activity; Fusion protein; Linker flexibility; Linker library