通过测定重组水蛭素Ⅲ的质粒中目的基因序列、重组水蛭素Ⅲ分子质量、C末端序列以及菌体蛋白和DNA残留量,对重组水蛭素Ⅲ的质量进行研究。结果:重组质粒经NheⅠ、HindⅢ双酶切和基因序列测定,表明工程菌所携带的质粒上含有完整的信号肽...通过测定重组水蛭素Ⅲ的质粒中目的基因序列、重组水蛭素Ⅲ分子质量、C末端序列以及菌体蛋白和DNA残留量,对重组水蛭素Ⅲ的质量进行研究。结果:重组质粒经NheⅠ、HindⅢ双酶切和基因序列测定,表明工程菌所携带的质粒上含有完整的信号肽基因、NheⅠ酶切位点、水蛭素HV3基因和HindⅢ酶切位点;还原性SDS PAGE结果表明在14 000 u处有单一条带,为重组水蛭素Ⅲ二聚体;测定样品分子C端序列为E D YA D E P I P E F,与重组水蛭素Ⅲ的理论值一致;重组水蛭素Ⅲ样品中的残留菌体蛋白和DNA含量分别是0.02%和每剂量样品中残留DNA含量低于2.8 ng。结论:从基因水平、蛋白水平和杂质的角度证明重组水蛭素Ⅲ产品符合质量控制标准。展开更多
cAMP receptor protein(CRP) plays profound roles in many bacteria as a global regulator.In Escherichia coli,CRP E.coli modulates the expression of many operons involved in carbon catabolism,in response to the fluctuati...cAMP receptor protein(CRP) plays profound roles in many bacteria as a global regulator.In Escherichia coli,CRP E.coli modulates the expression of many operons involved in carbon catabolism,in response to the fluctuation of intracellular cAMP level caused by carbon catabolism.A crp homologue gene has been identified in the genome of Pseudomonas putida,however,little is known about its cellular function.In this work,we investigated ligand response properties of this CRP protein(CRP P.putida).The results showed that in the presence of exogenous cAMP or cGMP,CRP P.putida can activate the lac promoter in E.coli cya crp mutant.In vitro isothermal titration calorimetry(ITC) assays indicated that CRP P.putida could bind cAMP as well as cGMP.Its affinity to cAMP is much higher than CRP E.coli.Sequence alignment of the CRP proteins suggested that the Thr132 of CRP P.putida(analogous to Ser128 of CRP E.coli) could be the key determinant for all ligand responsive properties observed above.When Thr132 of CRP P.putida is mutated to Serine,two phenomena were observed:(i) its affinity to cAMP or cGMP was reduced to a level similar to CRP E.coli ;(ii) its transcriptional activation activity on E.coli lac promoter was diminished.The potential physiological implications of these ligand binding properties are discussed.展开更多
文摘通过测定重组水蛭素Ⅲ的质粒中目的基因序列、重组水蛭素Ⅲ分子质量、C末端序列以及菌体蛋白和DNA残留量,对重组水蛭素Ⅲ的质量进行研究。结果:重组质粒经NheⅠ、HindⅢ双酶切和基因序列测定,表明工程菌所携带的质粒上含有完整的信号肽基因、NheⅠ酶切位点、水蛭素HV3基因和HindⅢ酶切位点;还原性SDS PAGE结果表明在14 000 u处有单一条带,为重组水蛭素Ⅲ二聚体;测定样品分子C端序列为E D YA D E P I P E F,与重组水蛭素Ⅲ的理论值一致;重组水蛭素Ⅲ样品中的残留菌体蛋白和DNA含量分别是0.02%和每剂量样品中残留DNA含量低于2.8 ng。结论:从基因水平、蛋白水平和杂质的角度证明重组水蛭素Ⅲ产品符合质量控制标准。
基金supported by the National Basic Research Program of China (2010CB126503)the National Natural Science Foundation of China (30830005 and 39925017)
文摘cAMP receptor protein(CRP) plays profound roles in many bacteria as a global regulator.In Escherichia coli,CRP E.coli modulates the expression of many operons involved in carbon catabolism,in response to the fluctuation of intracellular cAMP level caused by carbon catabolism.A crp homologue gene has been identified in the genome of Pseudomonas putida,however,little is known about its cellular function.In this work,we investigated ligand response properties of this CRP protein(CRP P.putida).The results showed that in the presence of exogenous cAMP or cGMP,CRP P.putida can activate the lac promoter in E.coli cya crp mutant.In vitro isothermal titration calorimetry(ITC) assays indicated that CRP P.putida could bind cAMP as well as cGMP.Its affinity to cAMP is much higher than CRP E.coli.Sequence alignment of the CRP proteins suggested that the Thr132 of CRP P.putida(analogous to Ser128 of CRP E.coli) could be the key determinant for all ligand responsive properties observed above.When Thr132 of CRP P.putida is mutated to Serine,two phenomena were observed:(i) its affinity to cAMP or cGMP was reduced to a level similar to CRP E.coli ;(ii) its transcriptional activation activity on E.coli lac promoter was diminished.The potential physiological implications of these ligand binding properties are discussed.
