Cloning and expression of Klebsiella phage K11 lysozyme gene.
Previously, the lysozyme gene of the Klebsiella phage K11 was partially sequenced in our lab. Using the sequence information the lysozyme gene of the Klebsiella phage K11 was amplified and cloned using the polymerase chain reaction of the pfu DNA polymerase. The nucleotide sequence of phage K11 lysozyme gene was determined. The open reading frame corresponds to a polypeptide with 151 amino acids and molecular weight of 16,932 Da. The deduced amino acid sequence of this polypeptide shows 74-75% homologies to the T7 and T3 phage lysozymes. Although the gene was efficiently expressed under the control of tac promoter in Escherichia coli XL1-blue cells at 37 degrees C, most of the K11 lysozyme produced was insoluble. When the temperature of cell growth was lowered, however, solubility of the K11 lysozyme was increased gradually. The insoluble protein expressed at 37 degrees C was solubilized in 5 M guanidine-HCl and refolded in the presence of oxido-shuffling agent (GSH/GSSG). Through the refolding process the recombinant lysozyme was solubilized and purified. The purified K11 lysozyme showed transcription inhibition of K11 RNA polymerase as well as amidase activity. These results showed that the lysozyme of bacteriophage K11 is a bifunctional protein that cuts a bond in the bacterial cell wall and selectively inhibits K11 phage RNA polymerase. Also, transcription inhibition ability of K11 lysozyme with T7 or SP6 phage RNA polymerase was measured. T7 RNA polymerase was less inhibited than K11 RNA polymerase by K11 lysozyme. But SP6 RNA polymerase was not nearly inhibited by K11 lysozyme.
Junn
Hyun Jung
HJ
Research Center for Bio-Medicinal Resources, Department of Life Science and Technology, Pai-Chai University, Daejeon, Republic of Korea.
Youn
Jooho
J
Suh
Kyong Hoon
KH
Lee
Sang Soo
SS
D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics; D002473:Cell Wall / Q000378:metabolism; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics*; D007709:Klebsiella / Q000821:virology*; D008969:Molecular Sequence Data; D009113:Muramidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic / Q000502:physiology
D011994:Recombinant Proteins; D012321:DNA-Directed RNA Polymerases; D009113:Muramidase; D000581:Amidohydrolases