• 한국축산식품학회지
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• 제39권4호
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• pp.601-609
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• 2019

Bifidobacterium longum KACC 91563 secretes family 5 extracellular solute-binding protein via extracellular vesicle. In our previous work, it was demonstrated that the protein effectively alleviated food allergy symptoms via mast cell specific apoptosis, and it has revealed a therapeutic potential of this protein in allergy treatment. In the present study, we cloned the gene encoding extracellular solute-binding protein of the strain into the histidine-tagged pET-28a(+) vector and transformed the resulting plasmid into the Escherichia coli strain BL21 (DE3). The histidine-tagged extracellular solute-binding protein expressed in the transformed cells was purified using Ni-NTA affinity column. To enhance the efficiency of the protein purification, three parameters were optimized; the host bacterial strain, the culturing and induction temperature, and the purification protocol. After the process, two liters of transformed culture produced 7.15 mg of the recombinant proteins. This is the first study describing the production of extracellular solute-binding protein of probiotic bacteria. Establishment of large-scale production strategy for the protein will further contribute to the development of functional foods and potential alternative treatments for allergies.

• Applied Biological Chemistry
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• 제31권4호
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• pp.382-386
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• 1988

Chemically defined medium을 사용하여 Bacillus sp. $T_2-3$으로 균체외단백질을 생산한 후 이들의 각종 물리화학적 성질을 조사 하였다. 균체외단백질은 전기영동과 gel 여과 결과로 분자량이 약 49,000이 되는 비슷한 2종의 단백질로 구성되어 있었다. 최대흡수파장은 230nm 부근이었으며 aspartic acid를 가장 많이 함유하고 있었다. 또한 균체외단백질의 물에대한 용해도는 55.8%, 0.4% NaOH에 대한 용해도는 28.4%로 albumin과 glutelin계통의 단백질로 생각되었다.

• 대한수의학회지
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• 제30권2호
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• pp.187-192
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• 1990

돼지로부터 분리한 Staphylococcus hyicus subsp hyicus 489주의 protein A 존재여부와 함량을 indirect hemagglutination 및 enzyme-linked immunosorbent assay(ELISA)법으로 조사하였다. Indirect hemagglutination text에 의하여 cell-bound protein A 및 extracellular protein A 보유균은 489주 중 각각 87.7% 및 36.0%로 나타났다. ELISA법에 의한 이들 균의 protein A함량 측정에서 전균주의 extracellular protein A는 1ng/ml미만이었으며, cell-bound protein A함량은 대부분의 균주에서 1ng/ml 미만이었고 11주가 25~108ng/ml 수준이었다.

• Applied Biological Chemistry
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• 제28권3호
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• pp.209-217
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• 1985

토양으로부터 균체외(菌體外) 단백질 생산균주 17주(株)를 분리하여 이 중 단백질생산이 강한 T219를 선정하여 동정(同定)하였으며 단백질 생산에 영향을 미치는 인자(因子)들을 검토하였다. T219균주(菌株)는 Bacillus속으로 동정(同定)되였으며 균체외(菌體外) 단백질생산 최적온도는 $25^{\circ}C$, pH는 7.5이었다. 단백질생산균주들이 분비하는 단백질에서는 protease와 amylase활성은 보이지 않았으며, 배양시간에 따른 단백질생산은 배양 2일에서 최대로 되었다. yeast extract와 meat extract를 함유한 배지에서 균체증식은 컸지만, 단백질 축적은 거의 없었고, polypeptone은 균체(菌體) 증식과 단백질생산에 큰 효과를 보였다. 또한 배지에 glycine과 L-isoleucine을 혼합하여 첨가하였을때 단백질생산의 효과가 커서 4mg/ml의 단백질축적이 있었다. T219균주(菌株)는 streptemycin의 $30{\mu}g/ml$이상의 농도에서 생육이 저해되었고, EDTA의 5mM농도에서는 생육에 영향을 받지 않았다. 또한, 이들 물질은 단백질생산에 영향을 미치지 않았다.

• Journal of Microbiology and Biotechnology
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• 제14권1호
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• pp.178-181
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• 2004

A gene encoding an extracellular nuclease was cloned from Aeromonas hydrophila strain ATCC14715. The gene was overexpressed and the enzyme was purified by fusing to maltose binding protein. It was shown that the protein possessed DNase activity on both single-stranded and double-stranded DNAs. It exhibited both endo- and exonuclease activities. It was also shown that the protein had an RNase activity. Possible roles of this extracellular enzyme in the A. hydrophila life cycle are discussed.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2000년도 Proceedings of 2000 KSAM International Symposium and Spring Meeting
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• pp.78-89
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• 2000

Protein secretion in filamentous fungi has been shown to be restricted to actively growing hyphal tips. To determine whether an increase in the amount of growing surface area of a fungus can lead to an increase in the amount of protein secretion, we examined secretion in a temperature-sensitive Neurospora crassa mcb mutant that shows a loss of growth polarity when incubated at restrictive-temperature. Incubation of the mcb mutant at restrictive-temperature results in a three- to five-fold increase in the level of extracellular protein and a 20- fold increase in carboxymethyl cellulase activity relative to a wild-type strain. A mutation in the cr-l gene has been shown previously to suppress the apolar growth phenotype of the mcb mutant, and we find that the level of extracellular protein produced by a mcb; cr-l double mutant was reduced to that of the wild-type control. Immunolocalization of a secreted endoglucanase revealed that proteins are secreted mainly at hyphal tips in hyphae exhibiting polar growth and over the entire surface area of bulbous regions of hyphae that are produced following a shift of the mcb mutant to restrictive-temperature. These results support the hypothesis that secretion of extracellular protein by a filamentous fungus can be significantly increased by mutations that alter growth polarity.

• Applied Biological Chemistry
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• 제31권2호
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• pp.187-192
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• 1988

미생물에 의한 단백질 생산과 그의 분비기작에 관한 기초 자료를 얻고자 토양으로 부터 균체외 단백질 생산균주를 분리하고 동정한 결과 Bacillus sp.로 추정 되었다. 선정균주를 3.0%의 glucose와 1.3%의 urea를 첨가한 chemically defined medium에 접종하여 $30^{\circ}C$에서 48시간 배양하였을때 1.2mg/ml의 단백질을 생산 하였고 $CaCO_3$의 첨가로 단백질 생산이 촉진 되었으며 $250{\mu}g/ml$의 cephalexin를 첨가하여 96시간 배양 하였을때 2.0mg/ml의 단백질을 생산 하였다.

• BMB Reports
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• 제49권11호
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• pp.585-586
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• 2016

Extracellular vesicles (EVs) are natural carriers of biomolecules that play central roles in cell-to-cell communications. Based on this, there have been various attempts to use EVs as therapeutic drug carriers. From chemical reagents to nucleic acids, various macromolecules were successfully loaded into EVs; however, loading of proteins with high molecular weight has been huddled with several problems. Purification of recombinant proteins is expensive and time consuming, and easily results in modification of proteins due to physical or chemical forces. Also, the loading efficiency of conventional methods is too low for most proteins. We have recently proposed a new method, the so-called exosomes for protein loading via optically reversible protein-protein interaction (EXPLORs), to overcome the limitations. Since EXPLORs are produced by actively loading of intracellular proteins into EVs using blue light without protein purification steps, we demonstrated that the EXPLOR technique significantly improves the loading and delivery efficiency of therapeutic proteins. In further in vitro and in vivo experiments, we demonstrate the potential of EXPLOR technology as a novel platform for biopharmaceuticals, by successful delivery of several functional proteins such as Cre recombinase, into the target cells.

• 한국미생물·생명공학회지
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• 제24권4호
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• pp.408-414
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• 1996

The SecY is a central component of the protein export machinery that mediate the translocation of secretory proteins across the plasma membrane, and has been known to be rate-limiting factor of secretion in Escherichia coli. In order to study the extracellular protein secretion in Gram-positive microorganism, we have, constructed strains harboring more than one copy of the gene for SecY. Firstly, the gene, for B. subtilis SecY and its promoter region was subcloned into pDH32 and the chimeric vector was inserted into amyE locus by homologous recombination. Secondly, low copy number vector, pCED6, was also used for subcloning the secY gene and for constructing a strain which harbors several copies of secY. The KH1 cell which harbor two copies of secY on the chromosome excreted more extracellular proteins than the wild type PB2. Moreover, the KH2 cells which harbor several copies of secY in pCED6 vector excreted more extracellular proteins than the KH1 cells. Here, we found that the capacity of protein secretion is partly controlled by the number of secY and it is suggested that SecY has also an important role in protein secretion in B. subtilis, a gram positive microorganism, as like in E. coli. This will promote the use of B. subtilis as a host for the expression of useful foreign gene and excretion of precious proteins.

• Archives of Pharmacal Research
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• 제21권3호
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• pp.315-319
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• 1998

Gastric smooth muscle of cats was used to investigate the involvement of protein kinase in vanadate-induced contraction. Vanadate caused a contraction of cat gastric smooth muscle in a dose-dependent manner. Vanadate-induced contraction was totally inhibited by 2 mM EGTA and 1.5 mM $LACI_3$ and significantly inhibited by $10\mu$M verapamil and $1\mu$M nifedipine, suggesting that vanadate-induced contraction is dependent on the extracellular $Ca^{2+}$ concentration, and the influx of extracellular $Ca^{2+}$ was mediated through voltage-dependent $Ca^{2+}$ channel. Both protein kinase C inhibitor and tyrosine kinase inhibitor significantly inhibited the vanadate-induced contraction and the combined inhibitory effect of two protein kinase inhibitors was greater than that of each one. But calmodulin antagonists did not have any influence on the vanadate-induced contraction. On the other hand, both forskolin ($1\mu$M) and sodium nitroprusside ($1\mu$M) significantly inhibited vanadate-induced contraction. Therefore, these results suggest that both protein kinase C and tyrosino kinase are involved in the vanadate-induced contraction which required the influx of extracellular $Ca^{2+}$ in cat gastric smooth muscle, and that the contractile mechanism of vanadate may be different from that of agonist binding to its specific receptor.