title: Overexpression of pucC improves the heterologous protein expression level in a Rhodobacter sphaeroides expression system.
abstract: The Rhodobacter sphaeroides system has been used to express membrane proteins. However, its low yield has substantially limited its application. In order to promote the protein expression capability of this system, the pucC gene, which plays a crucial role in assembling the R. sphaeroides light-harvesting 2 complex (LH2), was overexpressed. To build a pucC overexpression strain, a pucC overexpression vector was constructed and transformed into R. sphaeroides CQU68. The overexpression efficiency was evaluated by quantitative real-time polymerase chain reaction. A well-used reporter β-glucuronidase (GUS) was fusion-expressed with LH2 to evaluate the heterologous protein expression level. As a result, the cell culture and protein in the pucC overexpression strain showed much higher typical spectral absorption peaks at 800 and 850 nm compared with the non-overexpression strain, suggesting a higher expression level of LH2-GUS fusion protein in the pucC overexpression strain. This result was further confirmed by Western blot, which also showed a much higher level of heterologous protein expression in the pucC overexpression strain. We further compared GUS activity in pucC overexpression and non-overexpression strains, the results of which showed that GUS activity in the pucC overexpression strain was approximately ten-fold that in the non-overexpression strain. These results demonstrate that overexpressed pucC can promote heterologous protein expression levels in R. sphaeroides.
authors.affiliation: Key Laboratory of Biorheological Science and Technology Ministry of Education, Bioengineering College, Chongqing University, Chongqing, China.
authors.forename: L
authors.initials: L
authors.lastname: Cheng
authors.affiliation: Key Laboratory of Biorheological Science and Technology Ministry of Education, Bioengineering College, Chongqing University, Chongqing, China.
authors.forename: G
authors.initials: G
authors.lastname: Chen
authors.affiliation: Key Laboratory of Biorheological Science and Technology Ministry of Education, Bioengineering College, Chongqing University, Chongqing, China.
authors.forename: G
authors.initials: G
authors.lastname: Ding
authors.affiliation: Key Laboratory of Biorheological Science and Technology Ministry of Education, Bioengineering College, Chongqing University, Chongqing, China.
authors.forename: Z
authors.initials: Z
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Biorheological Science and Technology Ministry of Education, Bioengineering College, Chongqing University, Chongqing, China.
authors.forename: T
authors.initials: T
authors.lastname: Dong
authors.affiliation: Key Laboratory of Biorheological Science and Technology Ministry of Education, Bioengineering College, Chongqing University, Chongqing, China huzongli71@163.com.
authors.forename: Z
authors.initials: Z
authors.lastname: Hu
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D045332:Photosystem II Protein Complex / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D012242:Rhodobacter sphaeroides / Q000235:genetics* / Q000378:metabolism; D019076:Transgenes
chemical_list: D001426:Bacterial Proteins; D045332:Photosystem II Protein Complex; C100892:PucC protein, Rhodobacter; D011993:Recombinant Fusion Proteins; D005966:Glucuronidase