The amino acid sequences in the C-terminal region of glucose-1-phosphate thymidylyltransferases determine their soluble expression in Escherichia coli

Bijay Singh, Chang Beom Lee, Je Won Park, Jae Kyung Sohng

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)

Abstract

Two similar genes, dnmL and rmbA in Streptomyces peucetius, which encode for glucose-1-phosphate (G-1-P) thymidylyltransferases were expressed in Escherichia coli under similar conditions. While RmbA was expressed in soluble form, DnmL was found as insoluble aggregates in inclusion bodies. The difference in expression of these similar proteins led to investigate into the amino acid sequences of these proteins by sequence alignment, hydrophobicity scale and homology modeling. These analyses showed that the two proteins are different only in the C-terminal sequences. Deletion of C-terminal sequence of DnmL increased the expression level of truncated DnmL. Substitution of C-terminal sequence of DnmL with RmbA also expressed the recombinant protein in soluble form. Finally, mutation of six amino acids in DnmL rendered the protein expressed in soluble form. These results suggested that the soluble expression of the thymidylyltransferases lies in the C-terminal sequences. In conclusion, these methods of protein engineering will be a rational tool for enhancing solubility of proteins expressed in E.coli.

Original languageEnglish
Pages (from-to)179-187
Number of pages9
JournalProtein Engineering, Design and Selection
Volume25
Issue number4
DOIs
Publication statusPublished - 2012 Apr
Externally publishedYes

Bibliographical note

Funding Information:
This work was supported by the Intelligent Synthetic Biology Center of Global Frontier Project funded by the Ministry of Education, Science and Technology (2011-0031960) and a grant from the Next-Generation BioGreen 21 Program (SSAC, grant#: PJ008013), Rural Development Administration, Republic of Korea.

Keywords

  • hydrophobicity
  • inclusion bodies
  • recombinant DNA technology
  • thymidylyltransferase

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Molecular Biology

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