Bi-functional activities of chimeric lysozymes constructed by domain swapping between bacteriophage T7 and K11 lysozymes.
The lysozymes encoded by bacteriophage T7 and K11 are both bifunctional enzymes sharing an extensive sequence homology (75%). The constructions of chimeric lysozymes were carried out by swapping the N-terminal and C-terminal domains between phage T7 and K11 lysozymes. This technique generated two chimeras, T7K11-lysozyme (N-terminal T7 domain and C-terminal K11 domain) and K11T7-lysozyme (N-terminal K11 domain and C-terminal T7 domain), which are both enzymatically active. The amidase activity of T7K11-lysozyme is comparable with the parental enzymes while K11T7-lysozyme exhibits an activity that is approximately 45% greater than the wild-type lysozymes. Moreover, these chimeric constructs have optimum pH of 7.2-7.4 similar to the parental lysozymes but exhibit greater thermal stabilities. On the other hand, the chimeras inhibit transcription comparable with the parental lysozymes depending on the source of their N-terminals. Taken together, our results indicated that domain swapping technique localizes the N-terminal region as the domain responsible for the transcription inhibition specificity of the wild type T7 and K11 lysozymes. Furthermore, we were able to develop a simple and rapid purification scheme in purifying both the wild-type and chimeric lysozymes.
Alcantara
Ethel H
EH
Research Center for Bio-Medicinal Resources and Department of Life Science and Technology, Pai Chai University, 439-6 Doma-dong, Seo-gu, Daejeon 302-735, Korea.
Kim
Dong Hee
DH
Do
Su-Il
SI
Lee
Sang Soo
SS
D000581:Amidohydrolases / Q000378:metabolism; D017123:Bacteriophage T7 / Q000201:enzymology*; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005822:Genetic Vectors; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D009113:Muramidase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D009842:Oligopeptides; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics
C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011994:Recombinant Proteins; D014764:Viral Proteins; D006639:Histidine; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D009113:Muramidase; D000581:Amidohydrolases; C019665:amidase