• 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.

• Archives of Plastic Surgery
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• v.34 no.2
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• pp.141-148
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• 2007

Purpose: The object of this study was to evaluate the development of continuous osteogenic differentiation and bone formation after the subcutaneous implantation of the tissue-engineered bone, in vitro. Methods: Human adipose-derived stem cells were obtained by proteolytic digestion of liposuction aspirates. Adipose-derived stem cells were seeded in PLGA scaffolds after being labeled with PKH26 and cultured in osteogenic differentiation media for 1 month. The PLGA scaffolds with osteogenic stimulated adipose-derived stem cells were implanted in subcutaneous layer of four nude mice. Osteogenesis was assessed by RT-PCR for mRNA of osteopontin and bone sialoprotein(BSP), and immunohistochemistry for osteocalcin, and von Kossa staining for calcification of extracellular matrix at 1 and 2 months. Results: Implanted PLGA scaffold with adipose-derived stem cells were well vascularized, and PLGA scaffolds degraded and were substituted by host tissues. The mRNA of osteopontin and BSP was detected by RT-PCR in both osteogenic stimulation group and also osteocalcin was detected by immunohistochemistry at osteogenic stimulation 1 and 2 months, but no calcified extracellular deposit in von Kossa stain was found in all groups. Conclusion: In vivo, it could also maintain the characteristics of osteogenic differentiation that adipose-derived stem cells within PLGA scaffold after stimulation of osteogenic differentiation in vitro, but there were not normal bone formation in subcutaneous area. Another important factor to consider is in vivo, heterologous environment would have negative effect on bone formation as.[p1]

• Journal of Chest Surgery
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• v.45 no.5
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• pp.287-294
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• 2012

Background: Biologic valved grafts are important in cardiac surgery, and although several types of graft are currently available, most commercial xenografts tend to cause early disfiguration due to intimal proliferation and calcification. We studied the graft failure patterns on non-fixed and glutaraldehyde-fixed pulmonary xenograft in vivo animal experiment. Materials and Methods: Pulmonary valved conduits were obtained from the right ventricular outflow tract of eleven miniature pigs. The grafts were subjected to 2 different preservation methods; with or without glutaraldehyde fixation: glutaraldehyde fixation (n=7) and non-glutaraldehyde fixation (n=4). The processed explanted pulmonary valved grafts of miniature pig were then transplanted into eleven goats. Calcium quantization was achieved in all of the explanted xenograft, hemodynamic, histopathologic and radiologic evaluations were performed in the graft which the transplantation period was over 300 days (n=7). Results: Grafts treated with glutaraldehyde fixation had more calcification and conduit obstruction in mid-term period. Calcium deposition also appeared much higher in the glutaraldehyde treated graft compared to the non-glutaraldehyde treated graft (p<0.05). Conclusion: The present study suggests that xenografts prepared using glutaraldehyde fixation alone appeared to have severe calcification compared to the findings of non-glutaraldehyde treated xenografts and to be managed with proper anticalcification treatment and novel preservation methods. This experiment gives the useful basic chemical, histologic data of xenograft failure model with calcification for further animal study.

• Korean Journal of Plant Tissue Culture
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• v.27 no.3
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• pp.219-226
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• 2000

Lycopersicon peruvianum has a gametophytic self-incompatibility (GSI) mechanism controlled by a single genetic locus (S locus) with multiple alleles. S RNases, an allelic series of abundant stylar proteins, are products of the S locus in L. peruvianum and other Solanaceous plants. The $S_{11}$ RNase gene from L. peruvianum was introduced into a self-compatible (SC) species (Lycopersicon esculentum) to examine whether the expression pattern in the heterologous host mimics that in L. peruvianum. The resultant transgenic L. esculentum plants expressed the introduced gene highly in their styles, which is similar manner to the expresion in L. peruvianum. The $S_{11}$ RNase gene was expressed in the syle at a similar stage of flower development in both transgenic plants of L. esculentum and L. peruvianum without any morphological changes.

• Journal of Microbiology and Biotechnology
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• v.31 no.4
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• pp.570-583
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• 2021

Pyrococcus furiosus α-amylase can hydrolyze α-1,4 linkages in starch and related carbohydrates under hyperthermophilic condition (~ 100℃), showing great potential in a wide range of industrial applications, while its relatively low productivity from heterologous hosts has limited the industrial applications. Bacillus subtilis, a gram-positive bacterium, has been widely used in industrial production for its non-pathogenic and powerful secretory characteristics. This study was conducted to increase production of P. furiosus α-amylase in B. subtilis through three strategies. Initial experiments showed that co-expression of P. furiosus molecular chaperone peptidyl-prolyl cis-trans isomerase through genomic integration mode, using a CRISPR/Cas9 system, increased soluble amylase production. Therefore, considering that native P. furiosus α-amylase is produced within a hyperthermophilic environment and is highly thermostable, heat treatment of intact culture at 90℃ for 15 min was performed, thereby greatly increasing soluble amylase production. After optimization of the culture conditions (nitrogen source, carbon source, metal ion, temperature and pH), experiments in a 3-L fermenter yielded a soluble activity of 3,806.7 U/ml, which was 3.3- and 28.2-fold those of a control without heat treatment (1,155.1 U/ml) and an empty expression vector control (135.1 U/ml), respectively. This represents the highest P. furiosus α-amylase production reported to date and should promote innovation in the starch liquefaction process and related industrial productions. Meanwhile, heat treatment, which may promote folding of aggregated P. furiosus α-amylase into a soluble, active form through the transfer of kinetic energy, may be of general benefit when producing proteins from thermophilic archaea.

• Genes and Genomics
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• v.40 no.12
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• pp.1259-1267
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• 2018

Litchi (Litchi chinensis Sonn.) is an important subtropical fruit crop with high commercial value due to its high nutritional values and favorable tastes. However, irregular bearing attributed to unstable flowering is a major ongoing problem for litchi producers. Previous studies indicate that low-temperature is a key factor in litchi floral induction. In order to reveal the genetic and molecular mechanisms underlying the reproductive process in litchi, we had analyzed the transcriptome of buds before and after low-temperature induction using RNA-seq technology. A key flower bud differentiation associated gene, a homologue of FLORICAULA/LEAFY, was identified and named LcLFY (GenBank Accession No. KF008435). The cDNA sequence of LcLFY encodes a putative protein of 388 amino acids. To gain insight into the role of LcLFY, the temporal expression level of this gene was measured by real-time RT-PCR. LcLFY was highly expressed in flower buds and its expression correlated with the floral developmental stage. Heterologous expression of LcLFY in transgenic tobacco plants induced precocious flowering. Meantime, we investigated the sub-cellular localization of LcLFY. The LcLFY-Green fluorescent protein (GFP) fusion protein was found in the nucleus. The results suggest that LcLFY plays a pivotal role as a transcription factor in controlling the transition to flowering and in the development of floral organs in litchi.

• BMB Reports
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• v.52 no.7
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• pp.445-450
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• 2019

TTYH2 is a calcium-activated, inwardly rectifying anion channel that has been shown to be related to renal cancer and colon cancer. Based on the topological prediction, TTYH2 protein has five transmembrane domains with the extracellular N-terminus and the cytoplasmic C-terminus. In the present study, we identified a vesicle transport protein, ${\beta}$-COP, as a novel specific binding partner of TTYH2 by yeast two-hybrid screening using a human brain cDNA library with the C-terminal region of TTYH2 (TTYH2-C) as a bait. Using in vitro and in vivo binding assays, we confirmed the protein-protein interactions between TTYH2 and ${\beta}$-COP. We also found that the surface expression and activity of TTYH2 were decreased by co-expression with ${\beta}$-COP in the heterologous expression system. In addition, ${\beta}$-COP associated with TTYH2 in a native condition at a human colon cancer cell line, LoVo cells. The over-expression of ${\beta}$-COP in the LoVo cells led to a dramatic decrease in the surface expression and activity of endogenous TTYH2. Collectively, these data suggested that ${\beta}$-COP plays a critical role in the trafficking of the TTYH2 channel to the plasma membrane.

• Journal of Ginseng Research
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• v.43 no.4
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• pp.645-653
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• 2019

Background: Cytochrome P450 enzymes catalyze a wide range of reactions in plant metabolism. Besides their physiological functions on primary and secondary metabolites, P450s are also involved in herbicide detoxification via hydroxylation or dealkylation. Ginseng as a perennial plant offers more sustainable solutions to herbicide resistance. Methods: Tissue-specific gene expression and differentially modulated transcripts were monitored by quantitative real-time polymerase chain reaction. As a tool to evaluate the function of PgCYP736A12, the 35S promoter was used to overexpress the gene in Arabidopsis. Protein localization was visualized using confocal microscopy by tagging the fluorescent protein. Tolerance to herbicides was analyzed by growing seeds and seedlings on Murashige and Skoog medium containing chlorotoluron. Results: The expression of PgCYP736A12 was three-fold more in leaves compared with other tissues from two-year-old ginseng plants. Transcript levels were similarly upregulated by treatment with abscisic acid, hydrogen peroxide, and NaCl, the highest being with salicylic acid. Jasmonic acid treatment did not alter the mRNA levels of PgCYP736A12. Transgenic lines displayed slightly reduced plant height and were able to tolerate the herbicide chlorotoluron. Reduced stem elongation might be correlated with increased expression of genes involved in bioconversion of gibberellin to inactive forms. PgCYP736A12 protein localized to the cytoplasm and nucleus. Conclusion: PgCYP736A12 does not respond to the well-known secondary metabolite elicitor jasmonic acid, which suggests that it may not function in ginsenoside biosynthesis. Heterologous overexpression of PgCYP736A12 reveals that this gene is actually involved in herbicide metabolism.

• Journal of Microbiology and Biotechnology
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• v.29 no.3
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• pp.347-356
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• 2019

Bacillus sp. BS2 showing strong fibrinolytic activity was isolated from sea squirt (munggae) jeotgal, a traditional Korean fermented seafood. BS2 was identified as B. velezensis by molecular biological methods. B. velezensis BS2 grows well at 15% NaCl and at $10^{\circ}C$. When B. velezensis BS2 was cultivated in TSB broth for 96 h at $37^{\circ}C$, the culture showed the highest fibrinolytic activity ($131.15mU/{\mu}l$) at 96 h. Three bands of 27, 35 and 60 kDa were observed from culture supernatant by SDS-PAGE, and fibrin zymography showed that the major fibrinolytic protein was the 27 kDa band. The gene (aprEBS2) encoding the major fibrinolytic protein was cloned, and overexpressed in heterologous hosts, B. subtilis WB600 and E. coli BL21 (DE3). B. subtilis transformant showed 1.5-fold higher fibrinolytic activity than B. velezensis BS2. Overproduced AprEBS2 in E. coli was purified by affinity chromatography. The optimum pH and temperature were pH 8.0 and $37^{\circ}C$, respectively. $K_m$ and $V_{max}$ were 0.15 mM and $39.68{\mu}M/l/min$, respectively, when N-succinyl-Ala-Ala-Pro-Phe-pNA was used as the substrate. AprEBS2 has strong ${\alpha}$-fibrinogenase and moderate ${\beta}$-fibrinogenase activity. Considering its high fibrinolytic activity, significant salt tolerance, and ability to grow at $10^{\circ}C$, B. velezensis BS2 can be used as a starter for jeotgal.

• Journal of Microbiology and Biotechnology
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• v.31 no.6
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• pp.833-839
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• 2021

Bacillus subtilis CH3-5 isolated from cheonggukjang secretes a 28 kDa protease with a strong fibrinolytic activity. Its gene, aprE3-5, was cloned and expressed in a heterologous host (Jeong et al., 2007). In this study, the promoter of aprE3-5 was replaced with other stronger promoters (P_cry3A, P₁₀, P_SG1, P_srfA) of Bacillus spp. using PCR. The constructed chimeric genes were cloned into pHY300PLK vector, and then introduced into B. subtilis WB600. The P10 promoter conferred the highest fibrinolytic activity, i.e., 1.7-fold higher than that conferred by the original promoter. Overproduction of the 28 kDa protease was confirmed using SDS-PAGE and fibrin zymography. RT-qPCR analysis showed that aprE3-5 expression was 2.0-fold higher with the P10 promoter than with the original promoter. Change of the initiation codon from GTG to ATG further increased the fibrinolytic activity. The highest aprE3-5 expression was observed when two copies of the P₁₀ promoter were placed in tandem upstream of the ATG initiation codon. The construct with P10 promoter and ATG and the construct with two copies of P10 promoter in tandem and ATG exhibited 117% and 148% higher fibrinolytic activity, respectively, than that exhibited by the construct containing P10 promoter and GTG. These results confirmed that significant overproduction of a fibrinolytic enzyme can be achieved by suitable promoter modification, and this approach may have applications in the industrial production of AprE3-5 and related fibrinolytic enzymes.