• Archives of Pharmacal Research
• /
• 제14권3호
• /
• pp.261-265
• /
• 1991

In a microbial cell-free extract system, side chain cleavage on various sterols and steroids was tested. The cell-free extracts of Mycobacterium sp. NRRL B-3805 showed the side chain cleavage activity on lithocholic acid to form 5$\beta$-androstan-3.17-dione. The properties of the activity were examined.

• Journal of Industrial and Engineering Chemistry
• /
• 제67권
• /
• pp.231-235
• /
• 2018

In this study, we demonstrate the conversion of starch to (R,R)-2,3-butanediol (2,3-BD) in a hybrid cell-free synthesis system containing a mixture of lysates derived from Escherichia coli (E. coli) and cyanobacteria. A sufficient pool of pyruvate required for the synthesis of 2,3-BD was generated by combining metabolic pathways of cyanobacteria and E. coli. Successful synthesis of 2,3-BD was achieved by additional modifications of the hybrid cell-free system with the enzymes required to convert pyruvate to 2,3-BD. The results demonstrate a new approach to harness biological pathways to expand the scope of cell-free metabolic engineering by cross-species combinations of cell lysates.

• Molecules and Cells
• /
• 제37권6호
• /
• pp.473-479
• /
• 2014

Spermatogonial stem cells (SSCs, also called germline stem cells) are self-renewing unipotent stem cells that produce differentiating germ cells in the testis. SSCs can be isolated from the testis and cultured in vitro for long-term periods in the presence of feeder cells (often mouse embryonic fibroblasts). However, the maintenance of SSC feeder culture systems is tedious because preparation of feeder cells is needed at each subculture. In this study, we developed a Matrigel-based feeder-free culture system for long-term propagation of SSCs. Although several in vitro SSC culture systems without feeder cells have been previously described, our Matrigel-based feeder-free culture system is time- and cost-effective, and preserves self-renewability of SSCs. In addition, the growth rate of SSCs cultured using our newly developed system is equivalent to that in feeder cultures. We confirmed that the feeder-free cultured SSCs expressed germ cell markers both at the mRNA and protein levels. Furthermore, the functionality of feeder-free cultured SSCs was confirmed by their transplantation into germ cell-depleted mice. These results suggest that our newly developed feeder-free culture system provides a simple approach to maintaining SSCs in vitro and studying the basic biology of SSCs, including determination of their fate.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제11권3호
• /
• pp.258-261
• /
• 2006

The addition of zeolite particles enhances the stability of mRNA molecules in a cell-free protein synthesis system. When $20{\mu}g/{\mu}L$ of zeolite (Y5.4) is added to a reaction mixture of cell-free protein synthesis, a substantial increase in protein synthesis is observed. The stabilizing effect of zeolite is most dearly observed in an in vitro translation reaction directed by purified mRNA, as opposed to a coupled transcription and translation reaction. Upon the addition of zeolite in the in vitro translation reaction, the life span of the mRNA molecules is substantially extended, leading to an 80% increase in protein synthesis. The effect of zeolite upon the mRNA stability appears be strongly related to the cation exchange (potassium to sodium) reaction. Our results demonstrate the possibility of modifying this biological process using heterogeneous, non-biological substances in a cell-free protein synthesis system.

• 미생물학회지
• /
• 제8권1호
• /
• pp.35-40
• /
• 1970

In the cell-free systems of Dunaliella tertiolecta, fructosediphosphate aldolase hardly contribute to synthesize hexosephosphate from triosephosphate derived from pentosphosphate pathway, and it could be considered that glycerol synthesized from added glucose was synthesized but via 3-phosphoglyceraldehyde as an intermediate not hydroxypyruvate.

• 한국미생물·생명공학회지
• /
• 제26권3호
• /
• pp.195-199
• /
• 1998

Ethylene을 생성하는 호알칼리성 Bacillus sp. AIk-7를 분리 동정하였고, Bacillus sp. AIk-7의 ethylene 생성 경로와 전구 물질을 규명하기 위한 일환으로 이 균주의 intact cell과 cell-free system에서의 다양한 기질의 전환효과를 검토하였다. Intact cell과 cell-free system 모두에서 ethylene 생성을 극대화하기 위한 전환 조건은 3$0^{\circ}C$, pH 10.3으로 조사되었고, 기질 전환 효과를 검토한 결과 methionine(Met)과 1-amlnocyclopropane-1-carboxylic acid(ACC)가 압도적으로 많은 ethylene을 생성하였다. Cell-free system에서 저해제의 영향을 살펴본 결과 EDTA억제 효과로 2가 양이온이 필요함을, AOA 억제효과로 transaminase가 필요함을 알 수 있었다. Azide는 Met에서 ACC로의 단계에선 억제효과가 있었으나, ACC ethylene 전환 단계에선 오히려 활성효과를 보여주었다. 식물에서는 ACC에서 ethylene 과정은 Co$^{2+}$에 의해 저해받는데 반해 Bacillus sp. AIk-7은 Co$^{2+}$에 의해 ACC에서 ethylene과정이 10-70배 활성을 보였다. 이상의 결과를 통해 Bacillus sp. AIk-7에 의한 ethylene 생성 전구체는 Met과 ACC일 가능성이 있다.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제11권3호
• /
• pp.235-239
• /
• 2006

Due to recent advances in genome sequencing, there has been a dramatic increase in the quantity of genetic information, which has lead to an even greater demand for a faster, more parallel expression system. Therefore, interest in cell-free protein synthesis, as an alternative method for high-throughput gene expression, has been revived. In contrast to in vivo gene expression methods, cell-free protein synthesis provides a completely open system for direct access to the reaction conditions. We have developed an efficient cell-free protein synthesis system by optimizing the energy source and S30 extract. Under the optimized conditions, approximately $650{\mu}g/mL$ of protein was produced after 2h of incubation, with the developed system further modified for the efficient expression of PCR-amplified DNA. When the concentrations of DNA, magnesium, and amino acids were optimized for the production of PCR-based cell-free protein synthesis, the protein yield was comparable to that from the plasmid template.

• Korean Chemical Engineering Research
• /
• 제54권5호
• /
• pp.665-670
• /
• 2016

본 연구에서는 톨루엔 분해 균주인 Pseudomonas putida와 아세트산 분해 균주인 Cupriavidus necator에 무세포 효소 시스템(cell-free enzyme system)을 적용하여 톨루엔과 아세트산에 대한 분해 가능성을 확인하는 실험을 수행하였다. P. putida는 톨루엔 존재 하에서만 toluene dioxygenase를 생성하여 톨루엔을 cis-toluene dihydrodiol로 분해하며, C. necator는 acetyl coenzyme A synthetase-1을 생성하여 아세트산을 acetyl CoA로 전환시켜 생존에 필요한 ATP나 생분해성(biodegradable) 고분자인 Polyhydroxyalkanoate (PHA)를 합성한다. P. putida의 톨루엔 분해 효소인 toluene dioxygenase는 유도효소이기 때문에 toluene dioxygenase 생성 전과 후로 나누어 실험을 진행하였다. P. putida의 톨루엔 분해능력 확인을 위한 gas chromatography (GC) 분석 결과, 대조군과 toluene dioxygenase 생성 전인 실험군 1에서는 검출된 톨루엔의 양이 거의 유사하였으나, toluene dioxygenase 생성 후인 실험군 2에서는 검출된 톨루엔의 양이 대조군 및 실험군 1에 비해 감소하였다. 또한 C. necator의 아세트산 분해능력 확인을 위한 gas chromatography-mass spectrometer (GC-MS) 분석 결과, 무세포 효소 시스템을 적용한 실험군에서는 아세트산에 대한 피크가 검출되지 않았다. 따라서 P. putida와 C. necator는 무세포 효소 시스템 적용 후에도 톨루엔 및 아세트산 분해 능력이 유지되었으나, P. putida는 무세포 효소 시스템을 적용하기 전에 유도 효소를 생성하는 과정이 필요하다.

• 생명과학회지
• /
• 제15권5호
• /
• pp.779-782
• /
• 2005

Coupled transcription/translation cocktail을 이용하여 R. glutinis EH 유전자를 in vitro에서 합성하고 활성을 평가하였다. SDS-PAGE 및 immunoblotting을 통하여 45 kDa 크기의 EH 단백질이 발현되었음을 확인하였고, NBP assay 및 chiral GC 분석을 통해 발현된 단백질이 (R)-styrene oxide에 대한 입체선택성이 있음을 확인하였다. 따라서 무세포 단백질 합성 시스템을 이용하여 입체선택성을 유지시킨 EH 유전자 발현이 가능하며, 이러한 방법은 putative EH 유전자 탐색 등에 효율적으로 응용될 것이다.

• Journal of Plant Biology
• /
• 제12권2호
• /
• pp.7-14
• /
• 1969

Dunaliella tertiolecta did not show any increase in respiration rate when supplied with glucose, glycerol, sucrose, L-alanine, acetate, pyruvate and succinate. This was in contrast to Chlorella pyrenoidosa, which, under identical conditions, showed significant increase when supplied with glucose or acetate but not with the other compounds. Production of 14CO2 from added 14C-glucose in D. tertiolecta was lower than the other 14C-labelled substrates: L-alinine, glycerol, succinate, but higher than 14C-sucrose addition. And it was also lower than C. pyrenoidosa experiments which was added 14C-glucose as a substrate. Light reduced amounts of labelled carbon dioxide from 14C-glucose or 14C-acetate and increased incorporation of 14C from the substrates to cell materials in either D. tertiolecta or C. pyrenoidosa. The contribution of 14C from 14C-glucose to 14CO2 in cell-free system of D. tertiolecta were much higher than in whole cell suspension. It was contrast to C. pyrenoidosa which were showed reduction of 14CO2 production in cell-free systems than whole cell suspensions. When cell-free systems of D. tertiolecta and C. pyrenoidosa were supplied with ATP, NAD, NADP or/and hexokinase, it was remarkably increased production of 14CO2 from the substrates than the control. It was concluded that the low ability of D. tertiolecta to metabolize glucose were caused by the impermeability of the cell membrane to glucose and were not due to deficiencies of enzyme systems concerning glucose metabolism. In the cell-free systems, it seemed to be more active pentose phosphate pathway than glycolytic pathway in D. tertiolecta.