• Molecules and Cells
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• v.20 no.3
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• pp.361-363
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• 2005

Plasmid Achromobacter secretion (PAS) factor is a putative secretion factor that induces the secretion of periplasmic proteins. PAS factor from Vibrio vulnificus was crystallized at 294 K by the hanging drop vapor-diffusion method. It was isolated as a monomer during the purification procedures. The native crystal belongs to the F222 space group with unit cell parameters a = 56.1, b = 74.4, $c=80.0{\AA}$, ${\alpha}={\beta}={\gamma}=90^{\circ}$. The crystal was soaked in cryoprotectant containing 1 M NaBr for 1 h for MAD phasing. The diffraction limit of the Br-MAD data set was $1.9{\AA}$ using synchrotron X-ray irradiation at beam line BL-18B at the Photon Factory, Japan.

• Bulletin of the Korean Chemical Society
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• v.31 no.12
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• pp.3830-3833
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• 2010

• Journal of Microbiology and Biotechnology
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• v.17 no.8
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• pp.1316-1323
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• 2007

In order to induce high levels of protein secretion, we have constructed a recombinant plasmid, designated pBP244, into which was incorporated key components of the type-II See-dependent secretion system, including LepB (signal peptidase), SecA (ATPase), and SecB (chaperone). The biological activities of the LepB, SecA, and SecB components expressed from genes harbored by pBP244 appeared to play their normal roles. In order to evaluate the protein secretion, a pspA (Streptococcus $\underline{p}neumoniae\;\underline{s}urface\;\underline{p}rotein\;\underline{A}$) gene was cloned into pBP244, resulting in pBP438. S. typhimurium harboring pBP438 grown until the stationary phase, secreted a higher level of PspA into the culture supernatants than did the strain harboring pYA3494. The strain harboring pBP438 secreted a supernatant amount 1.71-fold, a periplasmic space amount 1.47-fold, and an outer membrane amount 1.49-fold higher than that of pYA3494. S. typhimurium ${\chi}8554$ kept the $Asd^+$ plasmid pBP244 and pBP438 for 60 generations in LB broth harboring DAP, thereby indicating that pBP244 and pBP438 were quite stable in the Salmonella strain.

• Journal of Life Science
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• v.14 no.3
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• pp.429-434
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• 2004

Levansucrase gene (levU) from Zymomonas mobilis was subcloned downstream of GALl promoter in pYES 2.0 and pYInu-AT [GALl0 promoter＋exoinulinase signal sequence of Kluyveromyces marxianus], resulting pYES-levU and pYInu-levU, respectively. The two expression plasmids were introduced into an invertase-deficient strain, Saccharomyces cerevisiae SEY2102, and then transformants showing high activity of levansucrase were selected. When each yeast transformants was cultivated in medium containing galactose, the extracellular and intracellular activities of levansucrase reached about 7.17 U/㎖ with the strain harboring pYES-levU and 6.61 U/㎖ with the strain harboring pYInu-levU. It was found that about 50% of levansucrase were detected in the medium and periplasmic space, and exoinulinase signal sequence didn't enhance the secretion efficiency. Furthermore, the recombinant levansucrase expressed in yeast seems to be produced as a hyper-glycosylated form.

• Microbiology and Biotechnology Letters
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• v.32 no.3
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• pp.206-211
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• 2004

Levansucrase gene(lscA) from Pseudomonas aurantiaca was subcloned downstream of GAL1 promoter in pYES 2.0 and pYInu-AT [GAL10 promoter ＋ exoinulinase signal sequence of Kluyveromyces marxianus], resulting pYES-lscA and p YInu-lscA, respectively. The two expression plasmids were introduced into an invertase-deficient strain, Sacchromayces cerevisiae SEY2102, and transformants with high activity of levansucrase were selected. When each yeast transform ants was cultivated in medium containing galactose, the extracellular and intracellular activities of levansucrase reached about 8.62 U/ml with the strain harboring pYES-lscA and 5.43 U/ml with the strain harboring pYInu-lscA. The levansucrase activity of 80% was detected in the periplasmic space and cytoplasm. The levansucrase activity in the medium of SEY2102/pYInu-lscA was 0.87 U/ml whereas that of SEY2102/pYES-lscA was 0.47 U/ml, which implying the exoinulinase signal sequence didn't enhance the secretion efficiency of levansucrase. Furthermore, the recombinant levansucrase expressed in yeast seems to be produced as a hyper-glycosylated form.

• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.72-72
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• 2003

The PAS factor, whose gene has been cloned from V vulnifcus, is a protein secretion factor. Although the role of the PAS factor in Vibrio is still unknown, it may be involved with the bacterial protein secretion. The PAS factor is a 76 amino acid polypeptide, and its expression in E. coli cells makes the host cell membrane leaky, resulting in the excretion of periplasmic proteins into the culture medium. Highly expressed PAS factor is harmful to the cell, this may be due to a disruption of the membrane structure or function.

• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.690-693
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• 2000

Efficient intron deletion with the correct splicing of the two exons of the human proinsulin gene was accomplished by a novel stepwise method using genomic DNA [5]. The two exons were separately amplified in two steps, using the second step primers that incorporated additional bases complementary to the other exon. The fragments were combined in a third PCR reaction. Cloning and sequencing of the PCR product demonstrated the correct splicing of the two exons. Expression studies, using the pET9a vector, revealed a protein band with the correct size with respect to human proinsulin as confirmed by SDS-PAGe and Western blot. Proinsulin concentration was estimated to be around 200 mg per liter culture, expressed as inclusion bodies. Protein secretion to the culture medium and periplasmic space was achieved by cloning in the pEZZ18 vector.

• KSBB Journal
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• v.18 no.4
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• pp.312-317
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• 2003

The addition of glycine up to 0.5％ (w/v) to Luria broth (LB) media on the secretion of levansucrase in a recombinant strain Escherichia coli JM109/pUPLK1 was observed to enhance the release of periplasmic proteins from the cell to the broth, without significantly affecting the cell growth rate and protein productivity. However, the glycine concentration at 1 ％ (w/v), the cell density attainable at the stationary phase fell to about 50％ and the extracellular activity of levansucrase corresponded to about 80％ of the total (extracellular plus intracellular) activity and increased by 2.6-fold, comparing to the cells grown in the absence of glycine. The increased pH at stationary phase accelerated the degradation of levansucrase. Maximal extracellular activity was attained when 1 ％ glycine was supplemented at the onset of strain growth.

• Journal of Life Science
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• v.27 no.12
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• pp.1403-1409
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• 2017

In this study, the xylitol dehydrogenase (XYL2) gene was expressed in Saccharomyces cerevisiae as a host cell for ease of use in the degradation of lignocellulosic biomass (xylose). To select suitable expression systems for the S.XYL2 gene from S. cerevisiae and the P.XYL2 gene from Pichia stipitis, $pGMF{\alpha}-S.XYL2$, $pGMF{\alpha}-P.XYL2$, $pAMF{\alpha}-S.XYL2$ and $pAMF{\alpha}-P.XYL2$ plasmids with the GAL10 promoter and ADH1 promoter, respectively, were constructed. The mating factor ${\alpha}$ ($MF{\alpha}$) signal sequence was also connected to each promoter to allow secretion. Each plasmid was transformed into S. cerevisiae $SEY2102{\Delta}trp1$ strain and the xylitol dehydrogenase activity was investigated. The GAL10 promoter proved more suitable than the ADH1 promoter for expression of the XYL2 gene, and the xylitol dehydrogenase activity from P. stipitis was twice that from S. cerevisiae. The xylitol dehydrogenase showed $NAD^+$-dependent activity and about 77% of the recombinant xylitol dehydrogenase was secreted into the periplasmic space of the $SEY2102{\Delta}trp1/pGMF{\alpha}-P.XYL2$ strain. The xylitol dehydrogenase activity was increased by up to 41% when a glucose/xylose mixture was supplied as a carbon source, rather than glucose alone. The expression system and culture conditions optimized in this study resulted in large amounts of xylitol dehydrogenase using S. cerevisiae as the host strain, indicating the potential of this expression system for use in bioethanol production and industrial applications.

• KSBB Journal
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• v.11 no.2
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• pp.165-172
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• 1996

To obtain a correctly folded human proinsulin, export of proinsulin using Staphylococcal protein A signal sequence-mediated secretion pathway has been attempted in E.coli. A secretion operon for proinsulin was constructed by consecutively connecting T7 promoter, SPA ribosome binding site, SPA signal sequence gene, and human proinsulin gene. Little immunoreactive proinsulin was detected in the periplasmic space and. culture medium, and not even in cytoplasmic space. The qualitative analysis of transcribed proinsulin mRNA and the in vitro transcription/translation experiment suggests that the negligible level of proinsulin export appears to be due to intracellular degradation of proinsulin, rather than due to the blockage during translocation. However, expression of proinsulin fusion protein such as MBP-proinsulin could dramatically increase export of proinsulin in E.coli.