• Journal of Microbiology and Biotechnology
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• 제9권2호
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• pp.196-200
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• 1999

A recombinant human $\alpha_{s1}$-casein was expressed as a secretory product in the yeast Saccharomyces cerevisiae. Three different leader sequences derived from the mating factor $\alpha$l (MF$\alpha$l), inulinase, and human $\alpha_{s1}$-casein were used to direct the secretion of human $\alpha_{s1}$-casein into the extracellular medium. Among the three leader sequences tested, the native leader sequence of human $\alpha_{s1}$-casein was found to be the most efficient in the secretory expression of human $\alpha_{s1}$-casein, which implies that the native leader sequence of human $\alpha_{s1}$-casein might be used very efficiently for the secretory production of other heterologous proteins in yeast. The recombinant human $\alpha_{s1}$-casein was proteolytically cleaved as the culture proceeded. Therefore, an attempt was made to produce human $\alpha_{s1}$-casein using a S. cerevisiae mutant in which the YAP3 gene encoding yeast aspartic protease 3 (YAP3) was disrupted. After 72 h of culture, most of the human $\alpha_{s1}$-casein secreted by the wild type was cleaved, whereas more than 70% of the human $\alpha_{s1}$-casein secreted by yap3-disruptant remained intact. The results suggest that YAP3 might be involved in the internal cleavage of human $\alpha_{s1}$-casein expressed in yeast

• 한국미생물·생명공학회지
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• 제19권3호
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• pp.215-221
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• 1991

YIp5를 사용하여 Cephalosporium acremonium ATCC 2033으로부터 Saccharomyces cerevisiae SHY3에서 발현되는 자율복제 기점 (ARS)를 분리하였다. 자율복제 기점을 갖는 40종의 vector 가운데 가장 안정성 (stability)이 높은 plasmid를 pCY-2라 명명하였으며 pCY-2는 C.acremonium에서 유래하는 3.7kb의 단편을 갖고 있었다. 또 Southern hybridization과 재형질전환을 통해 pCY-2가 효모내에서 자율적으로 복제되고 있다는 것을 확인하였다. 또한 pCY-2는 형질전환율과 안정성에 있어 효모의 ARS를 갖는 YRp7 vector보다 우수하였다.

• Journal of Microbiology and Biotechnology
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• 제6권6호
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• pp.451-455
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• 1996

Physiological studies on the expression of Bacillus thuringiensis subsp. tenebrionis (Btt) gene coding for insecticidal protein in recombinant Escherichia coli 537 were carried out to identify optimal culture condition. It was necessary to shift culture temperature from 30 to $42^{\circ}C$ to express the gene. Expression of the Btt toxin gene by recombinant E. coli 537 began within one hour after induction. Complex nitrogen sources increased production of the insecticidal protein. The total insecticidal protein was 0.5 g/I when using yeast extract as a complex nitrogen source. Soybean hydrolysate showed apparently the highest induction efficiency. After induction, the cellular content of the insecticidal protein was 5.4 times higher than it had been before induction. The optimal cultivation strategy was found to grow cells for 7hours at $30^{\circ}C$ and then 5-8 hours at $42^{\circ}C$. The optimal cultivation pH for the production of insecticidal protein was 6.5. The Btt toxin produced by the recombinant E. coli 537 was found to have the same level of potency against Colorado potato beetle as the original toxin.

• Journal of Microbiology and Biotechnology
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• 제9권2호
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• pp.179-183
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• 1999

A recombinant form of hirudin, a potent thrombin-specific inhibitor derived from the bloodsucking leech, was expressed as a secretory product in Saccharomyces cerevisiae under the control of GALl0 promoter and the mating factor $\alpha$pre-pro leader sequence. In an attempt to produce recombinant hirudin (r-Hir) of therapeutic purity in large quantities, the fed-batch fermentation was carried out by using this recombinant yeast, and subsequently downstream processing was developed with the preparative-scale column chromatography systems. About 234 mg/l of biologically active r-Hir was produced as a secretory product by the fed-batch fermentation strategy developed for an efficient downstream processing. Using a two-step chromatography process (an anion exchange chromatography followed by the reverse phase HPLC), the r-Hir was purified to>98% with an overall recovery yield of 84%. According to the N-terminal amino acid sequencing, the purified r-Hir was found to have the predicted N-terminal amino acid sequence. The biological activity of the purified r-Hir to inhibit thrombin was also identical to that of the commercial hirudin.

• Journal of Microbiology and Biotechnology
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• 제33권11호
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• pp.1513-1520
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• 2023

Kex2 protease (Kex2p) is a membrane-bound serine protease responsible for the proteolytic maturation of various secretory proteins by cleaving after dibasic residues in the late Golgi network. In this study, we present an application of Kex2p as an alternative endoprotease for the in vitro processing of recombinant fusion proteins produced by the yeast Saccharomyces cerevisiae. The proteins were expressed with a fusion partner connected by a Kex2p cleavage sequence for enhanced expression and easy purification. To avoid in vivo processing of fusion proteins by Kex2p during secretion and to guarantee efficient removal of the fusion partners by in vitro Kex2p processing, P₁', P₂', P₄, and P₃ sites of Kex2p cleavage sites were elaborately manipulated. The general use of Kex2p in recombinant protein production was confirmed using several recombinant proteins.

• 한국가금학회지
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• 제31권4호
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• pp.245-253
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• 2004

본 연구는 육계사료 내 rPST-yeast culture의 첨가 급여가 육계의 성장성적, 도체 특성 및 골격발달 등에 미치는 영향을 규명하기 위해 수행하였다. 2일령의 Ross 육용종 수평아리 460수를 공시하여 항생제, rPST-yeast culture 및 yeast culture가 첨가되지 않은 대조구 사료와 대조구 사료에 항생제(chlorotetracycline)만을 $0.1\%$ 수준으로 첨가한 실험사료(T1), rPST-yeast culture를 각각 $0.1\%$ 및 $0.2\%$ 수준으로 첨가한 실험사료(T2 및 T3) 및 yeast culture만을 $0.2\%$수준으로 첨가한 실험사료(T4)를 각각 6주간 급여하였다. 사료섭취량과 증체량은 주 단위로 조사하였다. 실험 5주차 종료 시에 각 처리구별로 10수씩 선발하여 도살하였고, 간, 비장, 복강지방 및 가슴근육의 상대적 중량과 가슴근육의 화학적 조성을 측정하였다. 혈액 내 GOT와 GPT 수치, 총 콜레스테롤, Ca 및 P을 측정하였으며, 경골의 중량, 파쇄 강도, 회분 함량, Ca및 P 함량을 측정하였다. 증체량은 대조구에 비하여 rPST-yeast culture를 $0.1\%$ 첨가한 실험구에서 증가하는 경향을 보였으며, 사료섭취 량 및 사료요구율은 처리구간 큰 차이가 없었다. 비장 및 복강 지방의 상대적 중량은 실험구간 유의한 차이가 관찰되지 않았으나, 간과 가슴근육의 상대적 중량은 처리간에 유의한 차이가 관찰되었는데,특히 T2처리구에서 가슴근육의 상대적 중량이 대조구에 비해 유의하게 증가하는 결과가 관찰되었다(p<0.05). 가슴 근육 내 조단백질 및 조지방 함량에 처리구간에 큰 차이는 없었으나, 수분 함량은 대조구에 비해 rPST-yeast culture와 SC yeast culture 첨가 급여구에서 유의하게 증가하였다(P<0.05). 혈청 내 GOT 수치는 변화가 없었으나, GPT수치는 rPST-yeast culture 첨가 급여 후에 유의하게 감소하는 결과가 얻어졌다(P<0.05). 혈중 총 콜레스테롤, Ca 및 P 농도는 처리간에 큰 차이를 보이지 않았다. 경골중량, 파쇄강도 및 화학적 성분 조성에서도 처리간에 큰 차이는 나타나지 않았다. 본 연구의 결과로부터 사료 내 rPST-yeast culture의 첨가급여에 의해 육계에서 성장성적이 향상될 가능성이 제시되었고, 특히 가슴근육이 증가와 같은 긍정적인 효과를 기대할 수 있을 것으로 사료되었다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.191-194
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• 2003

본 연구에서는 산업적으로 중요성이 날로 증가하고 있는 PLC의 대량 생산을 위해서 B. cereus ATCC10987에서 분리된 PLC 유전자를 pPICZa C에 삽입한 P. pastoris system을 개발하여 extracellular PLC를 생산하였다. 또한, 공업적 이용을 위해 여러 조건에서 B. cereus와 P. pastoris에서 생산된 extracellular PLC의 활성도를 측정하여 특성을 살펴보았다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVI)
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• pp.171-174
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• 2005

20 g/L peptone, 20 g/L dextrose, 10 g/L yeast extract에 100 mg/L zeocin을 첨가하여 동일하게 전배양 한 재조합 Pichia pastoris X-33/pBPT44를 각기 다른 탄소원이 든 배지에 배양하면서 12시간 간격으로 샘플을 채취하여 배양시간에 따른 세포성장, pH, 각 탄소원에 따른 PLC 생산량 등을 측정하였다.

• Biomolecules & Therapeutics
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• 제9권1호
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• pp.55-62
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• 2001

CJC-50100 is a recombinant hepatitis B virus surface antigen (HBsAg) expressed in yeast. The general pharmacological properties of CJC-50100 were evaluated in mice, rats, dogs and isolated guinea pig ileum. The doses were 0.33~33.3 $\mu$g/kg i.m. for mice and rats and 3.3~9.9 $\mu$g/kg i.v. for dogs. The concentrations of 0.002~0.02 $\mu$g/ml were used for the assay with guinea pig ileum. Intramuscular administration of CJC-50100 at the doses did not alter general behavior and the responses for central nervous system, smooth muscle, gastrointestinal system, cardiovascular and respiratory system, and water and electrolytes excretion. In summary, CJC-50100 had no pharmacological effect in these studies even up to the 100-fold of the expected clinical dose, 20 $\mu$g/man/60 kg.

• Journal of Microbiology and Biotechnology
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• 제10권4호
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• pp.564-567
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• 2000

A recombinant Saccharomyces cerevisiae, transformed with the genes encoding xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) orginated from Pichia stipitis CBS 5776, was developed to directly convert xylose to ethanol. A fed-batch fermentation with the recombinant yeast produced 8.7 g ethanol/l with a yield of 0.13 g ethanol/g xylose consumed.