A new technique was developed for the integrated processing of cell disruption and aqueous two-phase extraction in a high-speed bead mill to separate intracellular proteins from microbial cells. The process was named ...A new technique was developed for the integrated processing of cell disruption and aqueous two-phase extraction in a high-speed bead mill to separate intracellular proteins from microbial cells. The process was named as simultaneous cell disruption and aqueous two-phase extraction (SDATE). Advantages, such as high cell disruption efficiency, biochemical activities preservation of proteins, cell debris elimination, and prelimiary purification of the target protein were being claimed. When this technique was employed for isolating recombinant Tumor Necrosis Factor (TNF) from E. coli, overall protein concentration and TNF activity were found to have been increased. More than 95% of TNF was partitioned into the top phase and all cell debris were in the bottom phase. The partition coefficient was greater than 3 and the TNF purification factor was greater than 6. It is shown that less separation steps were being utilized in the new technique, meaning a reduction in separation time and less process extractors required.展开更多
Plasma membrane of plant cells is surrounded by cellulose wall and adjacent cells are joined together by a thick pectin rich matrix. Separation of plant cells and removal of the cell wall experimentally, by either a m...Plasma membrane of plant cells is surrounded by cellulose wall and adjacent cells are joined together by a thick pectin rich matrix. Separation of plant cells and removal of the cell wall experimentally, by either a mechanical or an enzymatic process, results in the production ofprotoplast. Protoplasts are useful tools to study the uptake and transport ofmacromolecules and production of somatic hybrids. Protoplasts can be obtained from all types of actively growing young and healthy tissues. The most convenient and widely used source of plant protoplasts is leaf. Juvenile seedling tissues, cotyledons are other alternative tissues most frequently used for protoplasts isolation. All the environmental and genotypic factors, which affect the cell wall thickenings and compactness indirectly, influence the number of protoplasts recovered. Protoplasts are isolated by two methods, mechanical and enzymatic. The enzyme mixture solution of celluiose/macerozyme is used to digest the cell wall. The critical factors affecting the obtaning ofprotoplasts are the kinds of cell wall degrading enzymes, the physiological state of plant leaves, the type of osmotic stabilizers and the composition of reaction solution. With the improvement of technique and enzyme combination rate, the yield of collected protoplasts will be increased higher.展开更多
基金Supported by the National Natural Science Foundation of China(No.295256O9 and 29736180).
文摘A new technique was developed for the integrated processing of cell disruption and aqueous two-phase extraction in a high-speed bead mill to separate intracellular proteins from microbial cells. The process was named as simultaneous cell disruption and aqueous two-phase extraction (SDATE). Advantages, such as high cell disruption efficiency, biochemical activities preservation of proteins, cell debris elimination, and prelimiary purification of the target protein were being claimed. When this technique was employed for isolating recombinant Tumor Necrosis Factor (TNF) from E. coli, overall protein concentration and TNF activity were found to have been increased. More than 95% of TNF was partitioned into the top phase and all cell debris were in the bottom phase. The partition coefficient was greater than 3 and the TNF purification factor was greater than 6. It is shown that less separation steps were being utilized in the new technique, meaning a reduction in separation time and less process extractors required.
文摘Plasma membrane of plant cells is surrounded by cellulose wall and adjacent cells are joined together by a thick pectin rich matrix. Separation of plant cells and removal of the cell wall experimentally, by either a mechanical or an enzymatic process, results in the production ofprotoplast. Protoplasts are useful tools to study the uptake and transport ofmacromolecules and production of somatic hybrids. Protoplasts can be obtained from all types of actively growing young and healthy tissues. The most convenient and widely used source of plant protoplasts is leaf. Juvenile seedling tissues, cotyledons are other alternative tissues most frequently used for protoplasts isolation. All the environmental and genotypic factors, which affect the cell wall thickenings and compactness indirectly, influence the number of protoplasts recovered. Protoplasts are isolated by two methods, mechanical and enzymatic. The enzyme mixture solution of celluiose/macerozyme is used to digest the cell wall. The critical factors affecting the obtaning ofprotoplasts are the kinds of cell wall degrading enzymes, the physiological state of plant leaves, the type of osmotic stabilizers and the composition of reaction solution. With the improvement of technique and enzyme combination rate, the yield of collected protoplasts will be increased higher.
