Human trefoil factor 2 （hTFF2） is considered as one of the most important initiators of mucosal healing in the gastrointestinal tract by promoting cell migration and suppressing apoptosis. However, it is hard to obt...Human trefoil factor 2 （hTFF2） is considered as one of the most important initiators of mucosal healing in the gastrointestinal tract by promoting cell migration and suppressing apoptosis. However, it is hard to obtain hTFF2 from human tissue and many recombinant hTFF2 produced in vitro exist as fusion proteins. The purpose of the present study was to produce native hTFF2 while maintaining its biological activities. The open reading frame of hTFF2 was inserted into a pET-32a（＋） expression vector, and hTFF2-TRX fusion protein was successfully expressed in Escherichia coli and purified by Nickel-nitrilotriacetic acid affinity chromatography and reverse-phase HPLC steps. The recombinant fusion protein （purity〉95%） was cleaved by Factor Xa at 23 ~C to release hTFF2. After removal of Factor Xa and undigested fusion proteins, hTFF2 was purified and identified by SDS-PAGE and Western blotting. The yield of recombinant hTFF2 was about 5 mg/L. The recombinant hTFF2 could promote IEC-6 cells migration and in vitro wound healing via the activation of ERK1/2. Recombinant hTFF2 could also inhibit apoptosis of HCT-116 cells induced by 50 lamol/L ceramide In summary, our results showed that the recombinant hTFF2 was expressed in E. coli and successfully purified after cleavage with the fusion partner with high yield while maintaining its biological activities. Recombinant hTFF2 might be useful for investigating the molecular mechanism of hTFF2 and development of hTFF2-related drugs.展开更多
OH-CATH is a novel cathelicidin identified from king cobra. It showed strong antibacterial activity against various bacteria in the presence of 1% NaCl and no haemolytic activity toward human red blood cells even at a...OH-CATH is a novel cathelicidin identified from king cobra. It showed strong antibacterial activity against various bacteria in the presence of 1% NaCl and no haemolytic activity toward human red blood cells even at a high concentration. OH-CATH might serve as model molecules for the development of antimicrobial drugs. Understanding the action mechanism of OH-CATH and the reason for its selectivity against microbes is very important for this purpose. The bactericidal effect of the king cobra antimicrobial peptide OH-CATH on Gram-negative Escherichia coli （ATCC 25922） is observed by scanning electron microscopy （SEM） and transmitted electron microscopy （TEM）. The SEM and TEM results suggested that the bactericidal mechanism of OH-CATH against Escherichia coli happened in three steps. Firstly, OH-CATH attached to the negatively charged bacterial wall by positively charged amino acid residues. In the second step, the accumulated OH-CATH aggregated and damaged the bacteria membrane in a pore-forming manner. In the last step, with the damage of cell permeability, the contents of the cells were released and eventually cells died.展开更多
基金"973"项目(2010CB529800)国家基金委面上项目(81160302,30870304)+1 种基金中国科学院"西部之光"(Y102291081)the Science and Technology Department of Yunnan Province (2011C1139)~~
文摘Human trefoil factor 2 （hTFF2） is considered as one of the most important initiators of mucosal healing in the gastrointestinal tract by promoting cell migration and suppressing apoptosis. However, it is hard to obtain hTFF2 from human tissue and many recombinant hTFF2 produced in vitro exist as fusion proteins. The purpose of the present study was to produce native hTFF2 while maintaining its biological activities. The open reading frame of hTFF2 was inserted into a pET-32a（＋） expression vector, and hTFF2-TRX fusion protein was successfully expressed in Escherichia coli and purified by Nickel-nitrilotriacetic acid affinity chromatography and reverse-phase HPLC steps. The recombinant fusion protein （purity〉95%） was cleaved by Factor Xa at 23 ~C to release hTFF2. After removal of Factor Xa and undigested fusion proteins, hTFF2 was purified and identified by SDS-PAGE and Western blotting. The yield of recombinant hTFF2 was about 5 mg/L. The recombinant hTFF2 could promote IEC-6 cells migration and in vitro wound healing via the activation of ERK1/2. Recombinant hTFF2 could also inhibit apoptosis of HCT-116 cells induced by 50 lamol/L ceramide In summary, our results showed that the recombinant hTFF2 was expressed in E. coli and successfully purified after cleavage with the fusion partner with high yield while maintaining its biological activities. Recombinant hTFF2 might be useful for investigating the molecular mechanism of hTFF2 and development of hTFF2-related drugs.
文摘OH-CATH is a novel cathelicidin identified from king cobra. It showed strong antibacterial activity against various bacteria in the presence of 1% NaCl and no haemolytic activity toward human red blood cells even at a high concentration. OH-CATH might serve as model molecules for the development of antimicrobial drugs. Understanding the action mechanism of OH-CATH and the reason for its selectivity against microbes is very important for this purpose. The bactericidal effect of the king cobra antimicrobial peptide OH-CATH on Gram-negative Escherichia coli （ATCC 25922） is observed by scanning electron microscopy （SEM） and transmitted electron microscopy （TEM）. The SEM and TEM results suggested that the bactericidal mechanism of OH-CATH against Escherichia coli happened in three steps. Firstly, OH-CATH attached to the negatively charged bacterial wall by positively charged amino acid residues. In the second step, the accumulated OH-CATH aggregated and damaged the bacteria membrane in a pore-forming manner. In the last step, with the damage of cell permeability, the contents of the cells were released and eventually cells died.
