Improving Solubility Of Shewanella Oneidensis MR-1 And Clostridium Thermocellum JW-20 Proteins Expressed Into Esherichia Coli

Authors

Irina Kataeva, University of Georgia
Jessie Chang, University of Georgia
Hao Xu, University of Georgia
Chi-Hao Luan, University of Alabama at Birmingham
Jizhong Zhou, Oak Ridge National Laboratory
Vladimir N. Uversky, Indiana University School of MedicineFollow
Dawei Lin, University of Georgia
Peter Horanyi, University of Georgia
Z. J. Liu, University of Georgia
Lars G. Ljungdahl, University of Georgia
John Rose, University of Georgia
Ming Luo, University of Alabama at Birmingham
Bi-Cheng Wang, University of Georgia

Document Type

Article

Publication Date

2005

Keywords

Gene Cloning and Expression, Improvement of Protein Solubility, Role of Fusions on Protein Solubility

Digital Object Identifier (DOI)

https://doi.org/10.1021/pr050108j

Abstract

Low solubility of proteins overexpressed in E. coli is a frequent problem in high-throughput structural genomics. To improve solubility of proteins from mesophilic Shewanella oneidensis MR-1 and thermophilic Clostridium thermocellum JW20, an approach was attempted that included a fusion of the target protein to a maltose-binding protein (MBP) and a decrease of induction temperature. The MBP was selected as the most efficient solubilizing carrier when compared to a glutathione S-transferase and a Nus A protein. A tobacco etch virus (TEV) protease recognition site was introduced between fused proteins using a double polymerase-chain reaction and four primers. In this way, 79 S. oneidensis proteins have been expressed in one case with an N-terminal 30-residue tag and in another case as a fusion protein with MBP. A foreign tag might significantly affect the properties of the target polypeptide. At 37 °C and 18 °C induction temperatures, only 5 and 17 tagged proteins were soluble, respectively. In fusion with MBP 4, 34, and 38 proteins were soluble upon induction at 37°, 28°, and 18 °C, respectively. The MBP is assumed to increase stability and solubility of a target protein by changing both the mechanism and the cooperativity of folding/unfolding. The 66 C. thermocellum proteins were expressed as fusion proteins with MBP. Induction at 37°, 28°, and 18 °C produced 34, 57, and 60 soluble proteins, respectively. The higher solubility of C. thermocellum proteins in comparison with the S. oneidensis proteins under similar conditions of induction correlates with the thermophilicity of the host. The two-factor Wilkinson−Harrison statistical model was used to identify soluble and insoluble proteins. Theoretical and experimental data showed good agreement for S. oneidensis proteins; however, the model failed to identify soluble/insoluble Clostridium proteins. A suggestion has been made that the Wilkinson−Harrison model is not applicable to C. thermocellum proteins because it did not account for the peculiarities of protein sequences from thermophiles.

Was this content written or created while at USF?

Yes

Citation / Publisher Attribution

Journal of Proteome Research, v. 4, issue 6, p. 1942-1951

Scholar Commons Citation

Kataeva, Irina; Chang, Jessie; Xu, Hao; Luan, Chi-Hao; Zhou, Jizhong; Uversky, Vladimir N.; Lin, Dawei; Horanyi, Peter; Liu, Z. J.; Ljungdahl, Lars G.; Rose, John; Luo, Ming; and Wang, Bi-Cheng, "Improving Solubility Of Shewanella Oneidensis MR-1 And Clostridium Thermocellum JW-20 Proteins Expressed Into Esherichia Coli" (2005). Molecular Medicine Faculty Publications. 752.
https://digitalcommons.usf.edu/mme_facpub/752