Jeong-Hoh Park

High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.

When expression of more than one gene is required in cells, bicistronic or multicistronic expression vectors have been used. Among various strategies employed to construct bicistronic or multicistronic vectors, an internal ribosomal entry site (IRES) has been widely used. Due to the large size and difference in expression levels between genes before and after IRES, however, a new strategy was required to replace IRES. A self-cleaving 2A peptide could be a good candidate to replace IRES because of its small size and high cleavage efficiency between genes upstream and downstream of the 2A peptide. Despite the advantages of the 2A peptides, its use is not widespread because (i) there are no publicly available cloning vectors harboring a 2A peptide gene and (ii) comprehensive comparison of cleavage efficiency among various 2A peptides reported to date has not been performed in different contexts. Here, we generated four expression plasmids each harboring different 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Thosea asigna virus and porcine teschovirus-1, respectively, and evaluated their cleavage efficiency in three commonly used human cell lines, zebrafish embryos and adult mice. Western blotting and confocal microscopic analyses revealed that among the four 2As, the one derived from porcine teschovirus-1 (P2A) has the highest cleavage efficiency in all the contexts examined. We anticipate that the 2A-harboring cloning vectors we generated and the highest efficiency of the P2A peptide we demonstrated would help biomedical researchers easily adopt the 2A technology when bicistronic or multicistronic expression is required.

Stage-Specific Expression of Pituitary Adenylate Cyclase-Activating Polypeptide Type I Receptor Messenger Ribonucleic Acid During Ovarian Follicle Development in the Rat1

Expression of pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide with considerable homology to vasoactive intestinal peptide, has been shown to be stimulated by gonadotropins in the ovary. The present studies further evaluated the cell-type specific expression and gonadotropin regulation of PACAP type I receptor (PACAPR) messenger RNA in immature rat ovaries and in cultured preovulatory follicles. Northern blot analysis of ovaries obtained from prepubertal rats revealed the increased expression of PACAPR during prepubertal development. The major cell types expressing PACAPR messenger RNA were granulosa cells of large preantral follicles. Treatment of immature rats with PMSG caused a decrease in ovarian PACAPR expression. In contrast, treatment with human (h) CG at 2 days after PMSG treatment stimulated ovarian PACAPR messenger RNA within 3–6 h in granulosa cells of preovulatory follicles. Treatment of cultured preovulatory follicles in vitro with LH further confirmed the time- and ...

Structures of iron-dependent alcohol dehydrogenase 2 from Zymomonas mobilis ZM4 with and without NAD+ cofactor.

The ethanologenic bacterium Zymomonas mobilis ZM4 is of special interest because it has a high ethanol yield. This is made possible by the two alcohol dehydrogenases (ADHs) present in Z. mobilis ZM4 (zmADHs), which shift the equilibrium of the reaction toward the synthesis of ethanol. They are metal-dependent enzymes: zinc for zmADH1 and iron for zmADH2. However, zmADH2 is inactivated by oxygen, thus implicating zmADH2 as the component of the cytosolic respiratory system in Z. mobilis. Here, we show crystal structures of zmADH2 in the form of an apo-enzyme and an NAD+–cofactor complex. The overall folding of the monomeric structure is very similar to those of other functionally related ADHs with structural variations around the probable substrate and NAD+ cofactor binding region. A dimeric structure is formed by the limited interactions between the two subunits with the bound NAD+ at the cleft formed along the domain interface. The catalytic iron ion binds near to the nicotinamide ring of NAD+, which is likely to restrict and locate the ethanol to the active site together with the oxidized Cys residue and several nonpolar bulky residues. The structures of the zmADH2 from the proficient ethanologenic bacterium Z. mobilis, with and without NAD+ cofactor, and modeling ethanol in the active site imply that there is a typical metal-dependent catalytic mechanism.

Crystal structure of Cmr5 from Pyrococcus furiosus and its functional implications

pfCmr5 and pfCmr4 bind by ion exchange chromatography (View interaction)

Structures of the γ‐class carbonic anhydrase homologue YrdA suggest a possible allosteric switch

The YrdA protein shows high sequence similarity to γ-class carbonic anhydrase (γ-CA) proteins and is classified as part of the γ-CA protein family. However, its function has not been fully elucidated as it lacks several of the conserved residues that are considered to be necessary for γ-CA catalysis. Interestingly, a homologue of γ-CA from Methanosarcina thermophila and a β-carboxysomal γ-CA from a β-cyanobacterium have shown that these catalytic residues are not always conserved in γ-CAs. The crystal structure of YrdA from Escherichia coli (ecYrdA) is reported here in two crystallographic forms. The overall structure of ecYrdA is also similar to those of the γ-CAs. One loop around the putative catalytic site shows a number of alternative conformations. A His residue (His70) on this loop coordinates with, or is reoriented from, the catalytic Zn(2+) ion; this is similar to the conformations mediated by an Asp residue on the catalytic loops of β-CA proteins. One Trp residue (Trp171) also adopts two alternative conformations that may be related to the spatial positions of the catalytic loop. Even though significant CA activity could not be detected using purified ecYrdA, these structural features have potential functional implications for γ-CA-related proteins.

Crystal structure of a Cas6 paralogous protein from Pyrococcus furiosus

Crystal structure of a Cas6 paralogous protein from Pyrococcus furiosus Hye-Mi Park, Minsang Shin, Jiali Sun, Gwang Sik Kim, Young Chul Lee, Jeong-Hoh Park, Bo Yeon Kim, and Jeong-Sun Kim* 1Department of Chemistry and Institute of Basic Sciences, Chonnam National University, 300, Yongbong-dong, Buk-gu, Gwangju 500-757, Korea 2Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, 300, Yongbong-dong, Buk-gu, Gwangju 500-757, Korea 3 Korea Research Institute of Bioscience and Biotechnology, 685-2, Yangcheongri, Cheong-Won, Choongbuk 363-883, Korea

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of an ASCH domain-containing protein from Zymomonas mobilis ZM4.

The human activating signal cointegrator 1 (ASC-1) homology (ASCH) domain is frequently observed in many organisms, although its function has not yet been clearly defined. In Zymomonas mobilis ZM4, the ZMO0922 gene encodes a polypeptide that includes an ASCH domain (zmASCH). To provide a better structural background for the probable role of ASCH domain-containing proteins, the ZMO0922 gene was cloned and expressed. The purified protein was crystallized from 30%(w/v) polyethylene glycol 400, 0.1 M cacodylic acid pH 6.5 and 0.2 M lithium sulfate. Diffraction data were collected to 2.1 Å resolution using synchrotron radiation. The crystal belonged to the primitive trigonal space group P3(1)21 or P3(2)21, with unit-cell parameters a=b=51.67, c=207.30 Å, α=β=90, γ=120°. Assuming the presence of one molecule in the asymmetric unit gave a Matthews coefficient of 4.69 Å(3) Da(-1), corresponding to a solvent content of 73.7%.