title: Improvement of expression level of polysaccharide lyases with new tag GAPDH in E. coli.
abstract: Escherichia coli (E. coli) is widely used to express a variety of heterologous proteins. Efforts have been made to enhance the expression level of the desired protein. However, problems still exist to regulate the level of protein expression and therefore, new strategies are needed to overcome those issues. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which is properly expressed in E. coli might play a leading role and increase the expression levels of the target proteins. In this study, GAPDH was fused with a target enzyme, ChSase ABC I, an endoeliminase and polysaceharide lyase. Our results confirmed this hypothesis and indicated that GAPDH boosted the expression level of ChSase ABC I with an increase of 2.25 times, while the enzymatic activity with an increase of 2.99 times. The hypothesis were also supported by RT-PCR study and GAPDH was more effective in enhancing the expression level and enzymatic activity as compared to MBP, which is commonly used as fused tag and can improve the soluble expression of target protein. addition, the expression level and enzymatic activity of other polysaceharide lyases were also improved in the presence of GAPDH. The findings of this study prove that GAPDH has a strong effect on enhancing the expression level and enzymatic activity of the target proteins.
authors: State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, No.15 East Road of North Third Ring, Chao Yang District, Beijing, 100029, China.
authors: Zhenya
authors: Z
authors: Chen
authors: Department of Biotechnology, Beijing Polytechnic, No.1 TaiyanggongShaoyaoju, Chao Yang District, Beijing, 100029, China. Electronic address: liyeyashi@163.com.
authors: Ye
authors: Y
authors: Li
authors: State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, No.15 East Road of North Third Ring, Chao Yang District, Beijing, 100029, China.
authors: Xinxiao
authors: X
authors: Sun
authors: State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, No.15 East Road of North Third Ring, Chao Yang District, Beijing, 100029, China. Electronic address: yuanqp@mail.buct.edu.cn.
authors: Qipeng
authors: Q
authors: Yuan
mesh_terms: D004926:Escherichia coli / Q000235:genetics*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D011133:Polysaccharide-Lyases / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D011133:Polysaccharide-Lyases
keywords: Activity; Expression level; GAPDH; Housekeeping gene