Development of a purification tag to produce thermostable fused protein.
- Author:
Weixin ZHAO
1
;
Song LIU
1
;
Liming LIU
1
;
Jian CHEN
1
;
Guocheng DU
1
Author Information
- Publication Type:Journal Article
- Keywords: expression host; fusion enzymes; production yield; purification; self-assembling amphipathic peptides; thermal stability
- MeSH: Escherichia coli; Green Fluorescent Proteins; Hydrophobic and Hydrophilic Interactions; Peptides; Pichia; Recombinant Fusion Proteins; metabolism
- From: Chinese Journal of Biotechnology 2019;35(4):626-635
- CountryChina
- Language:Chinese
- Abstract: Self-assembling amphipathic peptides (SAPs) have alternating hydrophilic and hydrophobic residues and can affect the thermal stabilities and catalytic properties of the fused enzymes. In this study, a novel multifunctional tag, S1vw (HNANARARHNANARARHNANARARHNARARAR) was developed to modify fused enzymes. After fusing S1vw at the enzymes/proteins N-terminus through a PT-linker, the crude enzymatic activities of polygalacturonate lyase and lipoxygenase were enhanced 3.1- and 1.89-fold, respectively, compared to the wild-type proteins. The relative fluorescence intensity of the green fluorescent protein was enhanced 16.22-fold. All the three S1vw fusions could be purified by nickel column with high purities and acceptable recovery rates. Moreover, S1vw also induced the thermostabilities enhancement of the fusions, with polygalacturonate lyase and lipoxygenase fusions exhibiting 2.16- and 3.2-fold increase compared with the corresponding wild-type, respectively. In addition, S1vw could enhance the production yield of green fluorescent protein in Escherichia coli and Bacillus subtilis while the production of GFP and its S1vw fusion changed slightly in Pichia pastoris. These results indicated that S1vw could be used as a multifunctional tag to benefit the production, thermal stability and purification of the fusion protein in prokaryotic expression system.
