title: Mutational analyses of Cry protein block7 polypeptides that facilitate the formation of protein inclusion in Escherichia coli.
abstract: 4AaCter is the polypeptide from the C-terminal extension of mosquitocidal Cry4Aa toxin, and facilitates formation of protein inclusion in Escherichia coli. It has been demonstrated that the use of 4AaCter as a peptide tag results in the efficient production of heterologous protein in E. coli. It has also been demonstrated that proteins are integrated, without losing their biological activities, into the protein inclusions. Although the mechanism to form protein inclusions in E. coli is unclear, highly conserved block7 sequence in 4AaCter is thought to be one of the functional factors. In this study, to analyze the ability of block7 to form protein inclusion, synthetic genes encoding the block7 polypeptide from selected 15 Cry proteins were constructed and expressed to produce glutathione S-transferase fusions in E. coli. Unexpectedly, only three of them (Cry5Ba, Cry32Aa, and Cry48Aa) formed protein inclusion as efficiently as that of Cry4Aa (>90% efficiency). The efficiencies in forming the protein inclusion were ranging from 39% to 66% for most of the tested block7s, and almost no protein inclusion was observed in Cry47Aa block7. This suggested that the ability of block7 to form the protein inclusion may vary with the type of Cry protein or the amino acid sequences. Mutational analyses revealed that substitution of the hydrophobic amino acids in block7 significantly affected the formation of protein inclusion, suggesting some important roles of these hydrophobic amino acid residues. Present results will contribute to develop a compact peptide tag based on block7 which forms the protein inclusion efficiently.
authors.affiliation: Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-Naka, Okayama, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Hayakawa
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Shimizu
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Ishida
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sakai
mesh_terms: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004731:Endotoxins / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000235:genetics* / Q000378:metabolism*; D002479:Inclusion Bodies / Q000378:metabolism*; D016296:Mutagenesis; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D050505:Mutant Proteins; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis