AIM: To identify a method for efficient large-scale purification of functional hepatitis B virus polymerase (HBV-Pol) without addition of cellular factors. METHODS: Full-length HBV-Pol (843 amino acids) tagged with 5&...AIM: To identify a method for efficient large-scale purification of functional hepatitis B virus polymerase (HBV-Pol) without addition of cellular factors. METHODS: Full-length HBV-Pol (843 amino acids) tagged with 5' end Polyhistidine was expressed at a high level in an Escherichia coli (E. coli ) system. Sodium dodecyl sulfate lysis buffer was utilized to dissolve insoluble HBV-Pol, and Ni-NTA resin affinity chromatography was utilized for HBV-Pol purification. Most recombinant HBV-Pol was eluted with 100 mmol/L imidazole in the presence of NP-40, a weak detergent that keeps HBV-Pol in solution. A reducing agent was utilized throughout the purification steps to keep soluble HBV-Pol from redundant disulfide bond formation. RESULTS: The large-scale production of functional intact human HBV-Pol was achieved in an E. coli expression system. Purified HBV-Pol showed stable reverse transcriptase activity and DNA polymerase activity. The purified protein was of high purity and had stable reverse transcriptase activity.CONCLUSION: Large-scale production of HBV-Pol in pure form should facilitate crystallization and detailed analysis of the structure and mechanism of HBV-Pol. Ability of this purification approach to obtain human HBV-Pol in an enzymatically active form should be helpful for development of drugs for treatment of chronic hepatitis B.展开更多
Brain and the gastrointestinal(GI)tract are intimately con-nected to form a bidirectional neurohumoral communica-tion system.The communication between gut and brain,knows as the gut-brain axis,is so well established t...Brain and the gastrointestinal(GI)tract are intimately con-nected to form a bidirectional neurohumoral communica-tion system.The communication between gut and brain,knows as the gut-brain axis,is so well established that the functional status of gut is always related to the condi-tion of brain.The researches on the gut-brain axis were traditionally focused on the psychological status affecting the function of the GI tract.However,recent evidences showed that gut microbiota communicates with the brain via the gut-brain axis to modulate brain development and behavioral phenotypes.These recent fi ndings on the new role of gut microbiota in the gut-brain axis implicate that gut microbiota could associate with brain functions as well as neurological diseases via the gut-brain axis.To elucidate the role of gut microbiota in the gut-brain axis,precise identification of the composition of microbes constituting gut microbiota is an essential step.However,identifi cation of microbes constituting gut microbiota has been the main technological challenge currently due to massive amount of intestinal microbes and the diffi culties in culture of gut microbes.Current methods for identifi ca-tion of microbes constituting gut microbiota are depend-ent on omics analysis methods by using advanced high tech equipment.Here,we review the association of gut microbiota with the gut-brain axis,including the pros and cons of the current high throughput methods for identi-fi cation of microbes constituting gut microbiota to eluci-date the role of gut microbiota in the gut-brain axis.展开更多
Recent advances in hematopoietic stem cells(HSCs)expansion by growth factors including angiopoietin-like proteins(Angptls)have opened up the possibility to use HSCs in regenerative medicine.However,the unavailability ...Recent advances in hematopoietic stem cells(HSCs)expansion by growth factors including angiopoietin-like proteins(Angptls)have opened up the possibility to use HSCs in regenerative medicine.However,the unavailability of true in vitro HSCs expansion by these growth factors has limited the understanding of the cellular and molecular mechanism of HSCs expansion.Here,we report the functional role of mouse Angptls 1,2,3,4,6 and 7 and growth factors SCF,TPO,IGF-2 and FGF-1 on purified mouse bone-marrow(BM)Lineage-Sca-1+(Lin-Sca-1+)HSCs.The recombinant retroviral transduced-CHO-S cells that secrete Angptls in serum-free medium were used alone or in combination with growth factors(SCF,TPO,IGF-2 and FGF-1).None of the Angptls stimu-lated HSC proliferation,enhanced or inhibited HSCs colony formation,but they did support the survival of HSCs.By contrast,any of the six Angptls together with saturating levels of growth factors dramatically stimulated a 3-to 4.5-fold net expansion of HSCs compared to stimulation with a combination of those growth factors alone.These findings lead to an understanding of the basic function of Angptls on signaling pathways for the survival as well as expansion of HSCs in the bone marrow niche.展开更多
基金Supported by Business for Cooperative R&D between Industry,Academy,and Research Institute funded Korea Small and Medium Business Administration in 2010,Grants No.08-1-28
文摘AIM: To identify a method for efficient large-scale purification of functional hepatitis B virus polymerase (HBV-Pol) without addition of cellular factors. METHODS: Full-length HBV-Pol (843 amino acids) tagged with 5' end Polyhistidine was expressed at a high level in an Escherichia coli (E. coli ) system. Sodium dodecyl sulfate lysis buffer was utilized to dissolve insoluble HBV-Pol, and Ni-NTA resin affinity chromatography was utilized for HBV-Pol purification. Most recombinant HBV-Pol was eluted with 100 mmol/L imidazole in the presence of NP-40, a weak detergent that keeps HBV-Pol in solution. A reducing agent was utilized throughout the purification steps to keep soluble HBV-Pol from redundant disulfide bond formation. RESULTS: The large-scale production of functional intact human HBV-Pol was achieved in an E. coli expression system. Purified HBV-Pol showed stable reverse transcriptase activity and DNA polymerase activity. The purified protein was of high purity and had stable reverse transcriptase activity.CONCLUSION: Large-scale production of HBV-Pol in pure form should facilitate crystallization and detailed analysis of the structure and mechanism of HBV-Pol. Ability of this purification approach to obtain human HBV-Pol in an enzymatically active form should be helpful for development of drugs for treatment of chronic hepatitis B.
文摘Brain and the gastrointestinal(GI)tract are intimately con-nected to form a bidirectional neurohumoral communica-tion system.The communication between gut and brain,knows as the gut-brain axis,is so well established that the functional status of gut is always related to the condi-tion of brain.The researches on the gut-brain axis were traditionally focused on the psychological status affecting the function of the GI tract.However,recent evidences showed that gut microbiota communicates with the brain via the gut-brain axis to modulate brain development and behavioral phenotypes.These recent fi ndings on the new role of gut microbiota in the gut-brain axis implicate that gut microbiota could associate with brain functions as well as neurological diseases via the gut-brain axis.To elucidate the role of gut microbiota in the gut-brain axis,precise identification of the composition of microbes constituting gut microbiota is an essential step.However,identifi cation of microbes constituting gut microbiota has been the main technological challenge currently due to massive amount of intestinal microbes and the diffi culties in culture of gut microbes.Current methods for identifi ca-tion of microbes constituting gut microbiota are depend-ent on omics analysis methods by using advanced high tech equipment.Here,we review the association of gut microbiota with the gut-brain axis,including the pros and cons of the current high throughput methods for identi-fi cation of microbes constituting gut microbiota to eluci-date the role of gut microbiota in the gut-brain axis.
基金supported by Technology Development Program for Agriculture and Forestry No.610002-3,Ministry for Food,Agriculture,Forestry and Fisheries,Republic of Korea。
文摘Recent advances in hematopoietic stem cells(HSCs)expansion by growth factors including angiopoietin-like proteins(Angptls)have opened up the possibility to use HSCs in regenerative medicine.However,the unavailability of true in vitro HSCs expansion by these growth factors has limited the understanding of the cellular and molecular mechanism of HSCs expansion.Here,we report the functional role of mouse Angptls 1,2,3,4,6 and 7 and growth factors SCF,TPO,IGF-2 and FGF-1 on purified mouse bone-marrow(BM)Lineage-Sca-1+(Lin-Sca-1+)HSCs.The recombinant retroviral transduced-CHO-S cells that secrete Angptls in serum-free medium were used alone or in combination with growth factors(SCF,TPO,IGF-2 and FGF-1).None of the Angptls stimu-lated HSC proliferation,enhanced or inhibited HSCs colony formation,but they did support the survival of HSCs.By contrast,any of the six Angptls together with saturating levels of growth factors dramatically stimulated a 3-to 4.5-fold net expansion of HSCs compared to stimulation with a combination of those growth factors alone.These findings lead to an understanding of the basic function of Angptls on signaling pathways for the survival as well as expansion of HSCs in the bone marrow niche.
