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Immunochemical Studies on Expression of Quinoproteins in Escherichia coli
RYOU, CHONGSUK,KIM, JAE-BEOM,KWON, MOOSIK 한국미생물 · 생명공학회 2000 Journal of microbiology and biotechnology Vol.10 No.1
An immunochemical method has been developed as the most sensitive tool for studying the expression of quinoproteins containing pyrroloquinoline quinone (PQQ) in E. coli. The PQQ was conjugated to bovine serum albumin (BSA), and the conjugant was purified by using a KwikSep^™ dextran desalting column chromatography. The PQQ-BSA conjugant was immunized to rabbits, and the IgG fractions of the antisera were purified. The most sensitive antibody againt PQQ-BSA conjugant recognized some nanogram quantity of the antigen on the blot, but had little cross reactivity with BSA. Using this batch of the antibody, all the immunochemical assays of quinoproteins in E. coli were performed. Some six different PQQ-specific spots were detected by Western blot analysis of the soluble proteins in E. coli after two-dimensional gel electrophoresis. Their molecular weights on the blot were estimated to be about 100-, 90-, 72-, 58-, 52-, and 50-kDa. Their pI values fell in the range from 4.8 to 5.5. These results strongly suggest that quinoproteins are present in E. coli, and that the protein moieties were covalently bound to PQQ.
A Gene Mutation of Human Dihydrolipoamide Dehydrogenase cDNA
Ryou, Chongsuk,Kwon, Moosik 성균관대학교 생명공학연구소 1999 生命工學硏究 Vol.5 No.1
Mammalian pyruvate dehydrogenase complex (PDC) catalyzes oxidative decarboxylation of pyruvate to produce Co2, acetyl-CoA, and NADH. Dihydrolipoamide Dehydrogenase (E3) is a component of the complex, and the enzymatic deficiency leads to lactic acidosis, increased concentrations of branched-chain amino acids in the plasma and increased urinary excretion of alpha-keto acids. The E3 deficiency also causes neurological degeneration due to the sensitivity of the central nervous system to defects in oxidative metabolism. In this study, an E3 mutant cDNA was generated from a patient showing some pyruvate metabolic defect. The mutant cDNA was subcloned into pBluescript SK-, and nested deletion set of the clone were prepared for the analysis of the whole nucleotide sequence. A silent mutation found in the clone meaning that there was no changes in amino acid sequence.
RYOU, CHONGSUK,CHUNG, TAEYOUNG,KWON, MOOSIK 한국미생물 · 생명공학회 2001 Journal of microbiology and biotechnology Vol.11 No.6
The gene coding for a Lepidoptera-specific insecticidal crystalline (or control) protein (ICP), recognized as cryLAc, from Bacillus thuringiensis subsp. kurstaki HD-73, was cloned into the vector pBluscript Ⅱ SK-, and then transformed in Escherichia coli DH5α. The clone was named EBtIAc and the chimeric phagemid, as pEBtIAc. Hyperexpression of CrylAc protoxin was observed in the extract of the culture of E. coli harboring pEBtIAc. Crystalline protoxin was purified by differential solubility. It was dissolved in alkaline pH, and exposed to trypsin to be activated. The molecular weights of the pro- and activated toxins on SDS-PAGE were estimated to be ca. 130 kDa and 60 kDa, respectively. The toxicity was tested by force-feeding larvae of gypsi moth (Lymantria dispar) with trypsinized protoxin. Using the batch of biologically active form of the toxin as an immunogen, anti-CrylAc antiserum was raised in a New Zealand white rabbit. Immunoglobulin G was fractionated from the serum by Protein-A sepharose affinity chromatography. Immunoreactivity of the antibody was examined by dot and Western blottings. It has been found that the anti-CryLAc antibody recognized the purified toxin at a level below a nanogram in terms of quantity. Using the antibody some of Bt-corns were able to be differentiated from tons of corn kernels which were imported from America as forage crops.
Gene Cloning and Nucleotide Sequence of Human Dihydrolipoamide Dehydrogenase-Binding Protein
Lee, Jeongmin,Ryou, Chongsuk,Jeon, Bong Kyun,Lee, Poongyeon,Woo, Hee-Jong,Kwon, Moosik Asian Australasian Association of Animal Productio 2002 Animal Bioscience Vol.15 No.3
The pyruvate dehydrogenase complex (PDC), a member of $\alpha$-keto acid dehydrogenase complex, catalyzes the oxidative decarboxylation of pyruvate with the formation of $CO_2$, acetyl-CoA, NADH, and $H^+$. This complex contains multiple copies of three catalytic components including pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3). Two regulatory components (E1-kinase and phospho-E1 phosphatase) and functionally less-understood protein (protein X, E3BP) are also involved in the formation of the complex. In this study, we have partially cloned the gene for E3BP in human. Nine putative clones were isolated by human genomic library screening with 1.35 kb fragment of E3BP cDNA as a probe. For investigation of cloned genes, Southern blot analysis and the construction of the restriction map were performed. One of the isolated clones, E3BP741, has a 3 kb-SacI fragment, which contains 200 bp region matched with E3BP cDNA sequences. The matched DNA sequence encodes the carboxyl-terminal portion of lipoyl-bearing domain and hinge region of human E3BP. Differences between yeast E3BP and mammalian E3BP coupled with the remarkable similarity between mammalian E2 and mammalian E3BP were confirmed from the comparison of the nucleotide sequence and the deduced amino acid sequence in the cloned E3BP. Cloning of human E3BP gene and analysis of the gene structure will facilitate the understanding of the role(s) of E3BP in mammalian PDC.
Targeting of poly(<small>l</small>-lysine) to organs that propagate prions
Lee, Hye-Mi,Ryou, Chongsuk SAGE Publications 2014 Journal of bioactive and compatible polymers Vol.29 No.5
<P>Poly(l-lysine) was recently discovered to inhibit prion propagation. To develop poly(l-lysine) as a potential therapeutic for prion diseases, the understanding of in vivo poly(l-lysine) behavior is pivotal. Therefore, to determine the poly(l-lysine) distribution in mouse spleen and brain, the primary and ultimate target organs for prions, we performed non-invasive longitudinal in vivo imaging and time course on live mice and ex vivo imaging on mouse organs to confirmed poly(l-lysine) was distributed, including the brain and spleen. By studying the in vivo and ex vivo fluorescence images, characteristic patterns of poly(l-lysine) accumulation and elimination depending on different administration routes were also found. Although only a portion of the administered poly(l-lysine) appears to target the brain and spleen, the specific poly(l-lysine) level in these organs was higher than that previously reported. Furthermore, the poly(l-lysine) retention in the brain and spleen was greater than that found in other organs. These results provide valuable information about poly(l-lysine) behavior in vivo, which will be an aid in designing optimal regimens for potential application of poly(l-lysine) in anti-prion therapeutics.</P>
Physiology of Cellular Prion Proteins in Reproduction
Zeljko M. Svedruzic,Chongsuk Ryou,Donchan Choi,Sung-Ho Lee,Yong-Pil Cheon The Korean Society of Developmental Biology 2024 발생과 생식 Vol.28 No.2
Cellular prion protein (PrP<sup>C</sup>) encoded at Prnp gene is well-known to form a misfolded isoform, termed scrapie PrP (PrP<sup>SC</sup>) that cause transmissible degenerative diseases in central nervous system. The physiological role of PrP<sup>C</sup> has been proposed by many studies, showing that PrP<sup>C</sup> interacts with various intracellular, membrane, and extracellular molecules including mitochondrial inner membrane as a scaffold. PrP<sup>C</sup> is expressed in most cell types including reproductive organs. Numerous studies using PrP<sup>C</sup> knockout rodent models found no obvious phenotypic changes, in particular the clear phenotypes in development and reproduction have not demonstrated in these knockout models. However, various roles of PrP<sup>C</sup> have been evaluated at the cellular levels. In this review, we summarized the known roles of PrP<sup>C</sup> in various cell types and tissues with a special emphasis on those involved in reproduction.