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Expression of a biologically active Ascaris suum cytochrome B5 in Escherichia coli
Oral Sessions I Parasitology
27. Molecular
international
EXPRESSION OFA BIOLOGICALLY ACl-IVE ASCARIS SL’UM CYTOCHROME BJ IN ESCHERICHIA COLI
Takamiva Department Medicine,
& Yamasaki H, Yu Y. Aoki T of Parasitology. Juntendo University Tokyo, 113-0033 Japan
School
O-0351
of
mammalian Ascoris suum cytochrome b5, like It erythrocyte cytochrome b5, is a soluble heme-protein. lacks the C-terminal membrane-anchoring domain that Our previous occurs in microsomal cytochromes bj. studies suggested that the nematode cytochrome b5 is synthesized as a precursor protein possessing an Nterminal presequence of 30 amino acids that is cleaved to form the mature protein comprising 82 amino acids. For studies on the structore and biosynthesis of A. suum nematode cylochrome b5 was cytochrome b5, the expressed in E. coli. previously A 563 bp cDNA encoding cytochrome b5, isolated from a” adult A. ~uurn cDNA library, was modified by PCR to generate a DNA sequence encoding a putative The cDNA was precursor protein of 112 amino acids. introduced into PET-28a(+) vector and expressed in E. coli strain BL21(DE3) using a” IPTG-inducible T7 RNA polymerase/promotor system. The transformed cells were red in color. Besides the they accumulated large insoluble precursor protein, amounts of soluble and processed cytochrome b5 with an N-terminal sequence identical to that of the native The processed protein purified from A. suum muscle. exhibited oxidized and reduced absorption cytochrome bj spectra identical to those of the “alive cytochrome b5. Furlhermore, the processed form was functionally active, and was reduced by NADH in like native cytochrome bg, the presence of A. suum microsomal fraction containing activity. These results clearly cytochrome b5 reductase functions presequence thal the N-terminal show physiologically in an E. coli expression system, providing and biologically active amounts of processed large prorein. MOLECULARCLONING AND EXPRESSION ENCODING
A
ERINACEIEUROPAEI
GROWTH
FACTOR
OF
OF
THE
GENE
SPIROMETRA
PLEROCERCOID
Uang flL, Ito K”. Konoda H”, Hori N”, Tanihata T’, Fukumoto Gaio K”. and Hirai X’ “Department of Molecular of Medical Zoology, *Department Faculty of Medicine, Tottori University, Yonago, Biology, Japan
Medical
and
-Department
University,
of
Laboratory
Sbi jiazhuang
Animal
050017,
Science,
People’s
Hebei
Republic
of
is
an
China. Spirometra
211
biology and Biochemistry-3
o-034')
O-0350
47 (Suppl.) (1998) 133-281
erinace1europaei
6.
erinaceieuropael)
intestinal tapeworm of wild and domesticated carnivores. Plerocercoids of this tapeworm have been shown to produce a kind of growth factor that can stimulate the growth of the infected mice (Phares and Hirai, 1990). Th’IS study is undertake” to clone the growth factor and investigate its expression in the worm. isolated from Materials and Yethods: nRNAs were plerocercoids of S. erinaceieuropaei (SEP) by a Pharmacia mRNA Isolation Kit and cDNA library was constructed with pSPORT 1 vector and screened by a probe isolated by PCR whose primers were designed according to the amino sequence of the identified protein that possesses both growth-stimulating function and cysteine proteinase activity. The fusion protein was produced in E. co/i by recloning the cDNA into the expression vector, pCEX5x-l. For Northern blotting, total RNAs were isolated by a” ISOCEN Kit, Results and Discussion: a 1.1.kb cDNA clone encoding this gene was isolated. Its sequence is different from those of the cysteine proteinases reported previously. The amino acid sequence predicted from the cloned gene comprises the fragment of the identified growth factor and has the catalytic site residues of cysteine protelnase. This gene expresses only i” SEP, not in the adult. The activity identification of the fusion protein and the effects of pH change, Cal’ removal, cyclohexiaide and actinomycin D on the growth dexaaethasone. factor gene expression will be present.
MOLECULAR CLONING AND CHARACTERIZATION OF CLONORCHIS SINENSIS ‘26 KDA AND 28 KDA GLUTATHIONE S-TRANSFERASE: A POTENT SERODIAGNOSTIC REAGENT
Shin-Yom Kang*, YiI-Yang Ah”*, Yang-Bae Chung*, Sung-Jong Hong*, Yoon Kong**,. Sang-YuIl Cho*+ *Department of Paramtology, College of Medicine, Chung-Ang University, Seoul 156-756, Korea, **Department of Molecular Parasitology, College of Medicine, Sung Kyun Kwan University, Suwon 440-746, Korea Glut&&me S-traosferases (GST) of trematodes are multifunctional enzymes neutralizii the endose”ous and exogenous free radicals and harmful xenobiitics. Trematode GSTs were proved to w immunogenic and to protect the immunized hosts from challenge infections. These findings prompted us to evaluate the antigenicity of Clonorchis sinewis GSTs. Wild 26 and 28 kDa GSTs were tied from adult C sine&s crude lysate through glutathione-sepharose afftity and DEAE anion exchange chmmatographies. The relative molar ratio of 28 kDa GST ta 26 kDa GST was about 2O:l. Specific activity of wild 26 kDa and 28 kDa GSTs using l-chloro-2,4 nitmbenzene was 86.7 and 150.0 unita’mg protein respectively. Inhibition of the two GSTs by family specific inhibitors was different each other. The 28 kDa GST was IoxIiied at the epithelial cells and contents of intestinal ceca, the tegument, and s-s in seminal vesicle and testes of the adult C. sinensis by immunohistozhemical staining usingt a monoclonal antibody (CsHK-17). cDNA fragments of C sinensis 26 kDa and 28 kDa GSTs were synthesized by PCR employing degenerated oliionucleotides and C sine&s total cDNA, and used as probes in screening a cDNA library constlucted in lammda ZAP II. A putative peptide (218 a.a.1 deduced from a 26 kDa GST cDNA clone showed 55.5-68.8% idendities to trematode 26 kDa GSTs. Its molecular weight was estimated by 25.1 kDa and by pl 6.03. A deduced peptide from a 28 kDa GST cDNA clone was estimated by molecular weight 24.5 kDa and by PI 8.95, and shuwed 37-45% amino acid identities to trematode 28 kDa GSTs. The mAb. CsHK-17, showed a positive reactivity to the recombinant ‘28GST but not to the recombinant 26 kDa
GST.
In
western
blotting,
C
sinensis
26
kDa
and 23 kDa
GSTs
revealed a wsitive reactivity to the C sine&s-infected human sera, but not to the sera from humans infected by Parogonimus westermi, Faxiota These results suggest C sinensu ‘26 hepatim or Schistmm mmsoni
kDn and 28 kDa GSTs useful semdiugnostic
ore cytosolic and secretory, and a cam&date of
reagent for clonorchiasis.
27. Molecular
international
EXPRESSION OFA BIOLOGICALLY ACl-IVE ASCARIS SL’UM CYTOCHROME BJ IN ESCHERICHIA COLI
Takamiva Department Medicine,
& Yamasaki H, Yu Y. Aoki T of Parasitology. Juntendo University Tokyo, 113-0033 Japan
School
O-0351
of
mammalian Ascoris suum cytochrome b5, like It erythrocyte cytochrome b5, is a soluble heme-protein. lacks the C-terminal membrane-anchoring domain that Our previous occurs in microsomal cytochromes bj. studies suggested that the nematode cytochrome b5 is synthesized as a precursor protein possessing an Nterminal presequence of 30 amino acids that is cleaved to form the mature protein comprising 82 amino acids. For studies on the structore and biosynthesis of A. suum nematode cylochrome b5 was cytochrome b5, the expressed in E. coli. previously A 563 bp cDNA encoding cytochrome b5, isolated from a” adult A. ~uurn cDNA library, was modified by PCR to generate a DNA sequence encoding a putative The cDNA was precursor protein of 112 amino acids. introduced into PET-28a(+) vector and expressed in E. coli strain BL21(DE3) using a” IPTG-inducible T7 RNA polymerase/promotor system. The transformed cells were red in color. Besides the they accumulated large insoluble precursor protein, amounts of soluble and processed cytochrome b5 with an N-terminal sequence identical to that of the native The processed protein purified from A. suum muscle. exhibited oxidized and reduced absorption cytochrome bj spectra identical to those of the “alive cytochrome b5. Furlhermore, the processed form was functionally active, and was reduced by NADH in like native cytochrome bg, the presence of A. suum microsomal fraction containing activity. These results clearly cytochrome b5 reductase functions presequence thal the N-terminal show physiologically in an E. coli expression system, providing and biologically active amounts of processed large prorein. MOLECULARCLONING AND EXPRESSION ENCODING
A
ERINACEIEUROPAEI
GROWTH
FACTOR
OF
OF
THE
GENE
SPIROMETRA
PLEROCERCOID
Uang flL, Ito K”. Konoda H”, Hori N”, Tanihata T’, Fukumoto Gaio K”. and Hirai X’ “Department of Molecular of Medical Zoology, *Department Faculty of Medicine, Tottori University, Yonago, Biology, Japan
Medical
and
-Department
University,
of
Laboratory
Sbi jiazhuang
Animal
050017,
Science,
People’s
Hebei
Republic
of
is
an
China. Spirometra
211
biology and Biochemistry-3
o-034')
O-0350
47 (Suppl.) (1998) 133-281
erinace1europaei
6.
erinaceieuropael)
intestinal tapeworm of wild and domesticated carnivores. Plerocercoids of this tapeworm have been shown to produce a kind of growth factor that can stimulate the growth of the infected mice (Phares and Hirai, 1990). Th’IS study is undertake” to clone the growth factor and investigate its expression in the worm. isolated from Materials and Yethods: nRNAs were plerocercoids of S. erinaceieuropaei (SEP) by a Pharmacia mRNA Isolation Kit and cDNA library was constructed with pSPORT 1 vector and screened by a probe isolated by PCR whose primers were designed according to the amino sequence of the identified protein that possesses both growth-stimulating function and cysteine proteinase activity. The fusion protein was produced in E. co/i by recloning the cDNA into the expression vector, pCEX5x-l. For Northern blotting, total RNAs were isolated by a” ISOCEN Kit, Results and Discussion: a 1.1.kb cDNA clone encoding this gene was isolated. Its sequence is different from those of the cysteine proteinases reported previously. The amino acid sequence predicted from the cloned gene comprises the fragment of the identified growth factor and has the catalytic site residues of cysteine protelnase. This gene expresses only i” SEP, not in the adult. The activity identification of the fusion protein and the effects of pH change, Cal’ removal, cyclohexiaide and actinomycin D on the growth dexaaethasone. factor gene expression will be present.
MOLECULAR CLONING AND CHARACTERIZATION OF CLONORCHIS SINENSIS ‘26 KDA AND 28 KDA GLUTATHIONE S-TRANSFERASE: A POTENT SERODIAGNOSTIC REAGENT
Shin-Yom Kang*, YiI-Yang Ah”*, Yang-Bae Chung*, Sung-Jong Hong*, Yoon Kong**,. Sang-YuIl Cho*+ *Department of Paramtology, College of Medicine, Chung-Ang University, Seoul 156-756, Korea, **Department of Molecular Parasitology, College of Medicine, Sung Kyun Kwan University, Suwon 440-746, Korea Glut&&me S-traosferases (GST) of trematodes are multifunctional enzymes neutralizii the endose”ous and exogenous free radicals and harmful xenobiitics. Trematode GSTs were proved to w immunogenic and to protect the immunized hosts from challenge infections. These findings prompted us to evaluate the antigenicity of Clonorchis sinewis GSTs. Wild 26 and 28 kDa GSTs were tied from adult C sine&s crude lysate through glutathione-sepharose afftity and DEAE anion exchange chmmatographies. The relative molar ratio of 28 kDa GST ta 26 kDa GST was about 2O:l. Specific activity of wild 26 kDa and 28 kDa GSTs using l-chloro-2,4 nitmbenzene was 86.7 and 150.0 unita’mg protein respectively. Inhibition of the two GSTs by family specific inhibitors was different each other. The 28 kDa GST was IoxIiied at the epithelial cells and contents of intestinal ceca, the tegument, and s-s in seminal vesicle and testes of the adult C. sinensis by immunohistozhemical staining usingt a monoclonal antibody (CsHK-17). cDNA fragments of C sinensis 26 kDa and 28 kDa GSTs were synthesized by PCR employing degenerated oliionucleotides and C sine&s total cDNA, and used as probes in screening a cDNA library constlucted in lammda ZAP II. A putative peptide (218 a.a.1 deduced from a 26 kDa GST cDNA clone showed 55.5-68.8% idendities to trematode 26 kDa GSTs. Its molecular weight was estimated by 25.1 kDa and by pl 6.03. A deduced peptide from a 28 kDa GST cDNA clone was estimated by molecular weight 24.5 kDa and by PI 8.95, and shuwed 37-45% amino acid identities to trematode 28 kDa GSTs. The mAb. CsHK-17, showed a positive reactivity to the recombinant ‘28GST but not to the recombinant 26 kDa
GST.
In
western
blotting,
C
sinensis
26
kDa
and 23 kDa
GSTs
revealed a wsitive reactivity to the C sine&s-infected human sera, but not to the sera from humans infected by Parogonimus westermi, Faxiota These results suggest C sinensu ‘26 hepatim or Schistmm mmsoni
kDn and 28 kDa GSTs useful semdiugnostic
ore cytosolic and secretory, and a cam&date of
reagent for clonorchiasis.