title: The amino acid sequences in the C-terminal region of glucose-1-phosphate thymidylyltransferases determine their soluble expression in Escherichia coli.
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.
authors.affiliation: Department of Pharmaceutical Engineering, Institute of Biomolecule Reconstruction, SunMoon University, #100, Kalsan-ri, Tangjeong-myeon, Asansi, Chungnam 336-708, Korea.
authors.forename: Bijay
authors.initials: B
authors.lastname: Singh
authors.forename: Chang Beom
authors.initials: CB
authors.lastname: Lee
authors.forename: Je Won
authors.initials: JW
authors.lastname: Park
authors.forename: Jae Kyung
authors.initials: JK
authors.lastname: Sohng
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005958:Glucosephosphates / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005958:Glucosephosphates; D011994:Recombinant Proteins; C031590:glucose-1-phosphate; D009713:Nucleotidyltransferases; C020587:glucose-1-phosphate thymidylyltransferase