Cloning, site-directed mutagenesis and expression of cathepsin L-like cysteine protease from Uronema marinum (Ciliophora: Scuticociliatida).
A cysteine protease gene (ScCtL) homologous to the cathepsin L genes was isolated from a cDNA library of the scuticociliate parasite (Uronema marinum). To express the ScCtL recombinant protein in heterologous system, 17 codons were redesigned to conform to the standard eukaryotic genetic code using PCR-based site-directed mutagenesis. The synthetic U. marinum procathepsin L (proScCtL) was expressed at high levels in E. coli BL21 (DE3) with pGEX-4T-1 vector, and successfully refolded and purified into a functional and enzymatically active form. The optimal pH for protease activity was found to be 4.5. Like any typical cysteine protease, the enzyme was inhibited by E-64 and leupeptin. A dot-blot immunoassay was conducted in an attempt to determine the reaction abilities and sensitivity of the anti-proScCtL polyclonal antibody to the cytosol and to the membrane fraction from the scuticociliate. Our results suggest that the biochemical characteristics of the recombinant ciliate proScCtL protein are similar to that of the cathepsin L-like cysteine protease, and that the PCR-based site-direct mutated ciliate gene was successfully expressed in a biochemically active form.
Ahn
Sang Jung
SJ
Department of Biotechnology, Pukyong National University, Busan 608-737, Republic of Korea.
Seo
Jung Soo
JS
Kim
Moo-Sang
MS
Jeon
Soo Jin
SJ
Kim
Na Young
NY
Jang
Jae Ho
JH
Kim
Ki Hong
KH
Hong
Yong-Ki
YK
Chung
Joon Ki
JK
Lee
Hyung Ho
HH
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016802:Oligohymenophorea / Q000201:enzymology* / Q000235:genetics; D017510:Protein Folding; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D015853:Cysteine Proteinase Inhibitors; D016054:DNA, Protozoan; D007976:Leupeptins; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; D007930:Leucine; C032854:leupeptin; C024974:E 64