An anti-metastatic polypeptide, bifunctional-domain (Cell Ⅰ -Hep Ⅱrecombinant polypeptide of human fibronectin. was expressed in E. coli and purified. The expression level was found to be about 20% 30 % of the tota...An anti-metastatic polypeptide, bifunctional-domain (Cell Ⅰ -Hep Ⅱrecombinant polypeptide of human fibronectin. was expressed in E. coli and purified. The expression level was found to be about 20% 30 % of the total cell proteins. In BL21 (DE3)/T7, an E. coli expressing system, lactose can be used as an inducer to substitute IPTG thereby reducing the cost by several hundredfold and it is suitable for the large-scale preparation of recombinant FN polypeptide. Cell Ⅰ -Hep Ⅱ fragment is an alkaline polypeptide. In BL21 (DE3)/T7 expressing system' better isolation was achieved if DEAE-52, instead of CM-52,was used for ion-exchange chromatography. The purified product was obtained after heparin-agarose affinity chromatography following ion-exchange chromatography.展开更多
Two ABA-specifically-inducible proteins from rice callus were isolated and purified by precipitation with 65-100% saturated （NH4）2SO4, followed by the DEAE-sepharose, TSK-gel, and two-dimension electrophoresis. Iso-...Two ABA-specifically-inducible proteins from rice callus were isolated and purified by precipitation with 65-100% saturated （NH4）2SO4, followed by the DEAE-sepharose, TSK-gel, and two-dimension electrophoresis. Iso-electric points （pl） of the proteins with the same molecular mass （24.5 kD） were 6.1 and 6.9, respectively. The Western blot analysis indicated that the proteins expressed in different tissues were obviously different. The A1 （pl 6.1） protein was only detected in calli treated with ABA and seed embryos （SE）. However, the A2 （pl 6.9） protein was found not only in the calli treated with ABA and SE, but also in the white dry callus. Thus it suggested that the two proteins might play some important roles in the processes of seed embryo （or somatic embryo） formation.展开更多
In order to construct the expression recombinant of human receptor associated protein （RAP）, optimize its expression condition and obtain the recombinant protein after expression with high efficiency, two prokaryoti...In order to construct the expression recombinant of human receptor associated protein （RAP）, optimize its expression condition and obtain the recombinant protein after expression with high efficiency, two prokaryotic expression vectors-pT7-PL and pET-28a（＋） were used to construct the expression recombinant containing RAP cDNA, and the expression efficiency of two kinds of expression E. coli of BL21 strains was compared. The effect of different induction conditions on the expression of recombinant RAP was observed. After recombinant protein was purified with Ni^＋ -nitrilotriacetic acid （Ni^＋ -NTA） affinity chromatogram, its binding ability with microphage was observed. The results showed that two recombinant plasmids both obtained high expression of RAP. The expression levels of RAP in plasmid pT7-PL-RAP in BL21 （DE3, plysS） strain were significantly higher than in BL21 （DE3） strain. The expression of pT7-PL-RAP in the presence of chloramphenicol was higher than in the absence of chloramphenicol, and most of the inducible expressed RAP was soluble. The RAP which was purified by Ni^＋ -NTA resin could strongly bind with the RAW264.7 cells rich in low density lipoprotein receptor （LDLR） family receptors. It was concluded that the expression condition of recombinant RAP was optimized and functional RAP was obtained, which offered a good foundation for the further production of RAP as research tool.展开更多
文摘An anti-metastatic polypeptide, bifunctional-domain (Cell Ⅰ -Hep Ⅱrecombinant polypeptide of human fibronectin. was expressed in E. coli and purified. The expression level was found to be about 20% 30 % of the total cell proteins. In BL21 (DE3)/T7, an E. coli expressing system, lactose can be used as an inducer to substitute IPTG thereby reducing the cost by several hundredfold and it is suitable for the large-scale preparation of recombinant FN polypeptide. Cell Ⅰ -Hep Ⅱ fragment is an alkaline polypeptide. In BL21 (DE3)/T7 expressing system' better isolation was achieved if DEAE-52, instead of CM-52,was used for ion-exchange chromatography. The purified product was obtained after heparin-agarose affinity chromatography following ion-exchange chromatography.
文摘Two ABA-specifically-inducible proteins from rice callus were isolated and purified by precipitation with 65-100% saturated （NH4）2SO4, followed by the DEAE-sepharose, TSK-gel, and two-dimension electrophoresis. Iso-electric points （pl） of the proteins with the same molecular mass （24.5 kD） were 6.1 and 6.9, respectively. The Western blot analysis indicated that the proteins expressed in different tissues were obviously different. The A1 （pl 6.1） protein was only detected in calli treated with ABA and seed embryos （SE）. However, the A2 （pl 6.9） protein was found not only in the calli treated with ABA and SE, but also in the white dry callus. Thus it suggested that the two proteins might play some important roles in the processes of seed embryo （or somatic embryo） formation.
基金This project was supported by a grant from National Natu-ral Sciences Foundation of China (No. 39970307).
文摘In order to construct the expression recombinant of human receptor associated protein （RAP）, optimize its expression condition and obtain the recombinant protein after expression with high efficiency, two prokaryotic expression vectors-pT7-PL and pET-28a（＋） were used to construct the expression recombinant containing RAP cDNA, and the expression efficiency of two kinds of expression E. coli of BL21 strains was compared. The effect of different induction conditions on the expression of recombinant RAP was observed. After recombinant protein was purified with Ni^＋ -nitrilotriacetic acid （Ni^＋ -NTA） affinity chromatogram, its binding ability with microphage was observed. The results showed that two recombinant plasmids both obtained high expression of RAP. The expression levels of RAP in plasmid pT7-PL-RAP in BL21 （DE3, plysS） strain were significantly higher than in BL21 （DE3） strain. The expression of pT7-PL-RAP in the presence of chloramphenicol was higher than in the absence of chloramphenicol, and most of the inducible expressed RAP was soluble. The RAP which was purified by Ni^＋ -NTA resin could strongly bind with the RAW264.7 cells rich in low density lipoprotein receptor （LDLR） family receptors. It was concluded that the expression condition of recombinant RAP was optimized and functional RAP was obtained, which offered a good foundation for the further production of RAP as research tool.
