• Molecules and Cells
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• v.26 no.1
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• pp.26-33
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• 2008

The plasmid pJB01, a member of the pMV158 family isolated from Enterococcus faecium JC1, contains three open reading frames, copA, repB, and repC. Plasmids included in this family produce counter-transcribed RNA (ctRNA) that contributes to copy number control. The pJB01 ctRNA, a transcript which consists of 54 nucleotides (nts), is encoded on the opposite strand from the copA/repB intergenic region and partially overlaps an atypical ribosome binding site (ARBS) for repB. The ARBS is integrated by the two underlined conserved regions: 5'-TTTTTGTNNNNTAANNNNNNNNNATG-3', and the ctRNA is complementary only to the 5' conserved sequence 5'-TTTTTGT-3'. This complementary sequence is located at a distance from the terminal loop of the ctRNA secondary structure. The ctRNA structure predicted by the mfold program suggests the possible generation of a terminal and an internal hairpin loop. The amount of in vitro translation product of repB mRNA was inversely proportional to the ctRNA concentration. Mutations in the terminal and internal hairpin loops of the ctRNA had inhibitory effects on its binding to the target mRNA. We propose that the intact structures of the terminal and internal hairpin loops, respectively, play important roles in forming the initial kissing and extending complexes between the ctRNA and target mRNA and that these regulate the copy number of this plasmid.

• Archives of Pharmacal Research
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• v.21 no.6
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• pp.712-717
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• 1998

The complete nucleotide sequence of the cDNA clone encoding rat skeletal muscle myosin- binding protein H (MyBP-H) was determined and amino acid sequence was deduced from the nucleotide sequence (GenBank accession number AF077338). The full-length cDNA of 1782 base pairs(bp) contains a single open reading frame of 1454 bp encoding a rat MyBP-H protein of the predicted molecular mass 52.7kDa and includes the common consensus 1CA__TG' protein binding motif. The cDNA sequence of rat MyBP-H show 92%, 84% and 41% homology with those of mouse, human and chicken, respectively. The protein contains tandem internal motifs array (-FN III-Ig C2-FN III- Ig C2-) in the C-terminal region which resembles to the immunoglobulin superfamily C2 and fibronectin type III motifs. The amino acid sequence of the C-terminal Ig C2 was highly conserved among MyBPs family and other thick filament binding proteins, suggesting that the C-terminal Ig C2 might play an important role in its function. All proteins belonging to MyBP-H member contains `RKPS` sequence which is assumed to be cAMP- and cGMP-dependent protein kinase A phosphorylation site. Computer analysis of the primary sequence of rat MyBP-H predicted 11 protein kinase C (PKC)phosphorylation site, 7 casein kinase II (CK2) phosphorylation site and 4N-myristoylation site.

• Journal of Microbiology and Biotechnology
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• v.24 no.3
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• pp.337-345
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• 2014

Pichia pastoris is one of the most widely used expression systems for the secretory expression of recombinant proteins. The secretory expression in P. pastoris usually makes use of the prepro $MAT{\alpha}$ sequence from Saccharomyces cerevisiae, which has a dibasic amino acid cleavage site at the end of the signal sequence. This is efficiently processed by Kex2 protease, resulting in the secretion of high levels of proteins to the medium. However, the proteins that are having the internal accessible dibasic amino acids such as KR and RR in the coding region cannot be expressed using this signal sequence, as the protein will be fragmented. We have identified a new signal sequence of 18 amino acids from a P. pastoris protein that can secrete proteins to the medium efficiently. The PMT1-gene-inactivated P. pastoris strain secretes a ~30 kDa protein into the extracellular medium. We have identified this protein by determining its N-terminal amino acid sequence. The protein secreted has four DDDK concatameric internal repeats. This protein was not secreted in the wild-type P. pastoris under normal culture conditions. We show that the 18-amino-acid signal peptide at the N-terminal of this protein is useful for secretion of heterologous proteins in Pichia.

• Korean Journal of Microbiology
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• v.38 no.2
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• pp.74-80
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• 2002

The purpose of this work was to investigate the characterization and sequence of catechol 1,2-dioxygenase (Cl,2O) purified from Acinetobacter sp. KS-1 which was grown on benzoate as a sole carbon source. Cl,2O demonstrated its enzyme activity to catechol and 4-methylcatechol. The optimum temperature of Cl,2O was $35^{\circ}C$, and the optimal pH was in the range from pH 7.5 to 9.0. $Ag^{+}$, $Hg^{+}$, and $Cu^{2+}$ showed inhibitory effect on the activity of Cl,2O. Molecular weight of the enzyme was determined to approximately 36 kDa by SDS-PAGE and 7-terminal amino acid sequence of Cl,2O was analyzed as $^{1}MNYQQIDALVKQMNVDTAKG^{20}$and exhibited 95% sequence homology with that of Cl,2O from Acinetobacter radioresistens In addition, trypsin digestion and peptide mapping were performed for internal sequencing analysis. Molecular weights of three digested peptide fragments were analyzed as 966.3 Da, 1933.8 Da and 2081.7 Da by MALDI-TOF, which were matched with each internal sequences $^{1}SQSDFNLRR^{9}\, ^{1}HGNRPSHVHYFNSAPGYR^{18}\, ^{1}TIEGPLYVAGAPESVGFAR^{19}$) of. A. radioresistens. PCR product was amplified with the degenerated primers derived from N-terminal and each internal amino acid sequences.

• Journal of Applied Biological Chemistry
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• v.42 no.2
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• pp.71-74
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• 1999

The crude enzyme prepared from the culture supernantant of Arthrobacter sp. S37 was purified by Phenyl Toyopearl column chromatography. Six endo-inulinases were detected by activity staining on native PAGE and named Inu I to Inu VI. Endo-inulinase were further purified by DEAE cellulose column chromatography and band slicing. Inu II~VI produced mainly inulotriose (F3) and inulotetraose (F4) as well as a small amount of inulobiose (F2) and fructose in contrast to Inu I producing F3, F4 and F5 from inulin. The N-terminal amino acid sequence of native and six CNBr-cleaved fragment of Inu VI were determined. No homology was found in amino acid sequences between Inu VI and other fructan hydrolase including invertase reported.

• Journal of Microbiology
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• v.35 no.3
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• pp.206-212
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• 1997

Heliobacillus mobilis is a strictly anaerobic Gram-positive bacterium which contains a primitive Photosystem I-type reaction center. The membrane-bound cytochrome $C_{553}$ from the heliobacterium suggested to be the immediate electron donor to the photooxidized pigment (P798+) has been isolated and characterized. The heme protein was visualized as a major component with an apparent molecular size of 17kDa in TMBZ-staining analysis of the membrane preparation and showed characteristic $\alpha$ (552.5 nm), $\beta$ (522nm), and Soret absorption (416 nm) peaks of a typical reduced c-type cytochrome in the partially purified sample. The internal 43 amino acid sequence of the electron donor was obtained by chemical agent and protease treatments followed by N-terminal sequencing of the resulting fragments. The internal sequence carries lots of lysine residues and a Cys-X-X-Cys-His sequence motif which are the characteristics of typical c-type cytochromes. The analysis of the sequence by FAST or FASTA program, however, did not show any significant similarity to other known heme proteins.

• BMB Reports
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• v.35 no.5
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• pp.524-531
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• 2002

Thrombotic thrombocytopenic purpura (TTP) is characterized by widespread platelet thrombi in arterioles and capillaries. Unusually large or multimeric von Willebrand factor, as well as one or ore platelet-agglutinating factors, have been implicated in the pathogenesis of TTP. But, the actual mechanisms of platelet agglutination have not been satisfactorily explained. Recent studies suggested the 37-kDa platelet-agglutinating protein (PAP) p37 to be partially responsible for the formation of platelet thrombi in patients with TTP. We studied mobility in SDS-PAGE, the sequence of N-terminal amino acid residues, DNA and antigenic characteristics of PAP p37, which might be related to the pathogenesis of TTP. PAP p37 was purified from the plasma of a 31-year-old male Korean patient with acute TTP. The findings are as follows: (1) We compared PAP p37 with thrombin through the use of SDS-PAGE, either with or without $\beta$-mercaptoethanol. PAP p37 did not appear to be cleaved between the A- and B-chains of prethrombin 2. However, thrombin did cleave between those of prethrombin 2, but linked with disulfide bridge. (2) N-terminal 21 amino acid sequence of PAP p37 was T-F-G-S-G-E-A-D-X-G-L-R-P-L-F-E-K-K-S-L-E. It appeared to be identical to that of 285-305 amino acid residues of human prothrombin (prethrombin 2). (3) No prothrombin gene DNA mutation was revealed. (4). The antigenicity of PAP p37 was similar to thrombin, which was a result of the competitive binding against the anti-thrombin antibody. With these results, we conclude that PAP p37 has similar characteristics to prethrombin2.

• Journal of Microbiology and Biotechnology
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• v.11 no.3
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• pp.508-514
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• 2001

A stuructural gene (ycl) encoding novel yeast cell wall hydrolase, YCL, was cloned from alkalophilic Bacillus alcalophilus subsp. YB380 by PCR, and transformed into E. coli JM83. Based on the N-terminal and internal amino acid sequences of the enzyme, primers were designed for PCr. The positive clone that harbors 1.8 kb of the yeast cell wall hydrolase gene was selected by the colony hybridization method with a PCR fragment as a probe. According to the computer analysis, this gene contained a 400-base-paired N-terminal domain of the enzyme. Based on nucletide homology of the cloned gene, a 850 bp fragment was amplified and the C-terminal domain of the enzyme was sequenced. With a combination of the two sequences, a full nucleotide sequence for YCL was obtained. This gene, ycl, consisted of 1,297 nucleotides with 27 nucleotides with 27 amino acids of signal sequence, 83 redundant amino acids of prosequence, and 265 amino acids of the mature protein. This gene was then cloned into the pJH27 shuttle vector and transformed into the Bacillus subtilis DB104 to express the enzyme. It was confirmed that the expressed cell wall hydrolase that was produced by Bacillus subtilis DB104 was the same as that of the donor strain, by Western blot using polyclonal antibody (IgY) prepared from White Leghorn hen. Purified yeast cell wall hydrolase and expressed recombinant protein showed a single band at the same position in the Western blot analysis.

• Journal of Microbiology and Biotechnology
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• v.30 no.1
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• pp.109-117
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• 2020

Cre recombinase is widely used to manipulate DNA sequences for both in vitro and in vivo research. Attachment of a trans-activator of transcription (TAT) sequence to Cre allows TAT-Cre to penetrate the cell membrane, and the addition of a nuclear localization signal (NLS) helps the enzyme to translocate into the nucleus. Since the yield of recombinant TAT-Cre is limited by formation of inclusion bodies, we hypothesized that the positively charged arginine-rich TAT sequence causes the inclusion body formation, whereas its neutralization by the addition of a negatively charged sequence improves solubility of the protein. To prove this, we neutralized the positively charged TAT sequence by proximally attaching a negatively charged poly-glutamate (E12) sequence. We found that the E12 tag improved the solubility and yield of E12-TAT-NLS-Cre (E12-TAT-Cre) compared with those of TAT-NLS-Cre (TAT-Cre) when expressed in E. coli. Furthermore, the growth of cells expressing E12-TAT-Cre was increased compared with that of the cells expressing TAT-Cre. Efficacy of the purified TAT-Cre was confirmed by a recombination test on a floxed plasmid in a cell-free system and 293 FT cells. Taken together, our results suggest that attachment of the E12 sequence to TAT-Cre improves its solubility during expression in E. coli (possibly by neutralizing the ionic-charge effects of the TAT sequence) and consequently increases the yield. This method can be applied to the production of transducible proteins for research and therapeutic purposes.

• BMB Reports
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• v.30 no.5
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• pp.342-345
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• 1997

Catechol 1,2-dioxygenase $I_1$ (CD $I_1$) gene of Acinetobacter Iwoffii K24, $catA_1$ was expressed in Escherichia coli and was partially purified by using a MonoQ column. Expressed CD $I_1$ had the same molecular weight as purified CD $I_1$ from A. Iwoffii K24 on SDS-PAGE. Expressed CD $I_1$ was also identified by Western blotting and peptide sequencing of N-terminal and internal regions. When compared with purified CD $I_1$ of A. Iwoffii K24, expressed CD $I_1$ had similar substrate specificities and the effects of compounds on enzyme activity. N-terminal amino acid sequence of CD I expressed in E. coli was the same as that of purified CD $I_1$, suggesting that CD $I_1$ may be under the same posttranslational processing in E. coli and A. Iwoffii K24.