It was widely thought that 3 variants of Kunitz type trypsin inhibitor(SBTi A 2) existed in soybean seed storage protein.Three of codominant alleles Ti a,Ti b and Ti c were identified to decode these SBTi A 2 inhibito...It was widely thought that 3 variants of Kunitz type trypsin inhibitor(SBTi A 2) existed in soybean seed storage protein.Three of codominant alleles Ti a,Ti b and Ti c were identified to decode these SBTi A 2 inhibitors and the amino acid sequences of them were determined,of which one or more different amino acid were found.Ti d was a new variant allele of SBTi A 2 discovered after analyzing more than 15 000 samples of soybean seed in China.In order to study the structure features of Ti d protein,the amino acid sequence of this protein was deduced from its coding region which was amplified by PCR from soybean genomic DNA and sequenced.By comparison with Ti a protein,two different amino acid residue were found between Ti a and Ti d proteins.One was an extra Ala inserted in the signal peptide of Ti d,with 8 residues from N terminal,and the other existed in the mature protein,with Glu 69 in Ti a protein turned into Lys 69 in Ti d protein.The amino acid sequence of Ti d mature protein was also different from those of Ti a and Ti b.展开更多
The light chain of inter-α inhibitor, also known as bikunin or urinary trypsin inhibitor, is composed of two tandemly arranged Kunitz-type protease inhibitor domains. The second domain of bikunin has factor Xa inhibi...The light chain of inter-α inhibitor, also known as bikunin or urinary trypsin inhibitor, is composed of two tandemly arranged Kunitz-type protease inhibitor domains. The second domain of bikunin has factor Xa inhibitory activity which previously was enhanced by mutating two amino acids, glutamine 19 and tyrosine 46 to lysine and aspartate, respectively. In this study, we tried to potentiate its inhibitory activity against leukocyte elastase. A molecular docking model of the second domain of bikunin with leukocyte elastase revealed that P5 arginine 11 was a candidate residue for a third substitution. We generated six triple point mutants using site-directed mutagenesis, compared their leukocyte elastase-inhibitory activities, and selected the most potent variant with arginine 11 substituted to serine. The IC50 values for factor XIa, factor Xa, and leukocyte elastase were 182, 302, and 273 nM, respectively. Moreover, this triple point mutant prolonged the activated partial thromboplastin time and moderately reduced leukocyte elastase-induced endothelial injury. Additionally, favorable conformations created by these mutations were speculated using the structure of the Kunitz protease inhibitor domain of protease nexin 2 complexed with factor XIa as a reference. We discovered a novel triple point mutant of the second domain of bikunin that has potent inhibitory activities against factor XIa, factor Xa, and leukocyte elastase. This variant exhibited anticoagulant activity in plasma and suppressed endothelial cell injury.展开更多
目的分离美洲钩虫(Necator americanus)Kunitz型丝氨酸蛋白酶抑制剂1(Na Ku I1)c DNA,并进行原核表达,研究其抑制蛋白酶的效果。方法根据Gen Bank上预测的不完整的Na Ku I基因序列(XM_013449790)设计引物,运用快速扩增c DNA末端技术(SMA...目的分离美洲钩虫(Necator americanus)Kunitz型丝氨酸蛋白酶抑制剂1(Na Ku I1)c DNA,并进行原核表达,研究其抑制蛋白酶的效果。方法根据Gen Bank上预测的不完整的Na Ku I基因序列(XM_013449790)设计引物,运用快速扩增c DNA末端技术(SMART-RACE)分别从美洲钩虫成虫c DNA中扩增Na Ku I1 c DNA的5′和3′末端序列,拼接获得全长Na Ku I1 c DNA。将Na Ku I1成熟肽编码基因连接入原核表达载体,构建重组质粒p ET32a-sumo/Na Ku I1,转化至大肠埃希菌(Escherichia coli)BL21(DE3)中,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导Na Ku I1融合蛋白表达。表达产物包涵体经变性、复性、Ni-NTA亲和层析纯化后,用SUMO蛋白酶切割融合蛋白标签后获得重组蛋白r Na Ku I1。用凝血时间法检测r Na Ku I1的抗凝活性,发色底物法检测其对人纤溶酶、胰蛋白酶、中性粒细胞弹性蛋白酶、组织蛋白酶G和蛋白酶3、猪胰蛋白酶和胰弹性蛋白酶及牛α-胰糜蛋白酶的抑制作用。结果获得Na Ku I1全长c DNA,其编码的多肽由84个氨基酸残基组成,其中含16个氨基酸残基组成的信号肽,68个氨基酸残基组成的成熟肽。成熟肽原核表达产物为不可溶的包涵体。经变性、复性后纯化的r Na Ku I1无抗凝血活性。在100倍摩尔浓度比下,r Na Ku I1对人纤溶酶(5 nmol/L)、人胰蛋白酶(1 nmol/L)和猪胰蛋白酶(5 nmol/L)活性的抑制率近100%,对牛α-胰糜蛋白酶(1 nmol/L)和人中性粒细胞弹性蛋白酶(5 nmol/L)活性的抑制率分别约31.45%和25.18%,对人组织蛋白酶G、蛋白酶3和猪胰弹性蛋白酶均无抑制作用。r Na Ku I1抑制人胰蛋白酶及纤溶酶的抑制常数(ki)分别为(21.17±7.22)和(21.72±3.95)nmol/L。结论成功分离获得Na Ku I1全长c DNA序列,其原核表达产物r Na Ku I1具有较强抑制胰蛋白酶和纤溶酶活性的特点。展开更多
文摘It was widely thought that 3 variants of Kunitz type trypsin inhibitor(SBTi A 2) existed in soybean seed storage protein.Three of codominant alleles Ti a,Ti b and Ti c were identified to decode these SBTi A 2 inhibitors and the amino acid sequences of them were determined,of which one or more different amino acid were found.Ti d was a new variant allele of SBTi A 2 discovered after analyzing more than 15 000 samples of soybean seed in China.In order to study the structure features of Ti d protein,the amino acid sequence of this protein was deduced from its coding region which was amplified by PCR from soybean genomic DNA and sequenced.By comparison with Ti a protein,two different amino acid residue were found between Ti a and Ti d proteins.One was an extra Ala inserted in the signal peptide of Ti d,with 8 residues from N terminal,and the other existed in the mature protein,with Glu 69 in Ti a protein turned into Lys 69 in Ti d protein.The amino acid sequence of Ti d mature protein was also different from those of Ti a and Ti b.
文摘The light chain of inter-α inhibitor, also known as bikunin or urinary trypsin inhibitor, is composed of two tandemly arranged Kunitz-type protease inhibitor domains. The second domain of bikunin has factor Xa inhibitory activity which previously was enhanced by mutating two amino acids, glutamine 19 and tyrosine 46 to lysine and aspartate, respectively. In this study, we tried to potentiate its inhibitory activity against leukocyte elastase. A molecular docking model of the second domain of bikunin with leukocyte elastase revealed that P5 arginine 11 was a candidate residue for a third substitution. We generated six triple point mutants using site-directed mutagenesis, compared their leukocyte elastase-inhibitory activities, and selected the most potent variant with arginine 11 substituted to serine. The IC50 values for factor XIa, factor Xa, and leukocyte elastase were 182, 302, and 273 nM, respectively. Moreover, this triple point mutant prolonged the activated partial thromboplastin time and moderately reduced leukocyte elastase-induced endothelial injury. Additionally, favorable conformations created by these mutations were speculated using the structure of the Kunitz protease inhibitor domain of protease nexin 2 complexed with factor XIa as a reference. We discovered a novel triple point mutant of the second domain of bikunin that has potent inhibitory activities against factor XIa, factor Xa, and leukocyte elastase. This variant exhibited anticoagulant activity in plasma and suppressed endothelial cell injury.
文摘目的分离美洲钩虫(Necator americanus)Kunitz型丝氨酸蛋白酶抑制剂1(Na Ku I1)c DNA,并进行原核表达,研究其抑制蛋白酶的效果。方法根据Gen Bank上预测的不完整的Na Ku I基因序列(XM_013449790)设计引物,运用快速扩增c DNA末端技术(SMART-RACE)分别从美洲钩虫成虫c DNA中扩增Na Ku I1 c DNA的5′和3′末端序列,拼接获得全长Na Ku I1 c DNA。将Na Ku I1成熟肽编码基因连接入原核表达载体,构建重组质粒p ET32a-sumo/Na Ku I1,转化至大肠埃希菌(Escherichia coli)BL21(DE3)中,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导Na Ku I1融合蛋白表达。表达产物包涵体经变性、复性、Ni-NTA亲和层析纯化后,用SUMO蛋白酶切割融合蛋白标签后获得重组蛋白r Na Ku I1。用凝血时间法检测r Na Ku I1的抗凝活性,发色底物法检测其对人纤溶酶、胰蛋白酶、中性粒细胞弹性蛋白酶、组织蛋白酶G和蛋白酶3、猪胰蛋白酶和胰弹性蛋白酶及牛α-胰糜蛋白酶的抑制作用。结果获得Na Ku I1全长c DNA,其编码的多肽由84个氨基酸残基组成,其中含16个氨基酸残基组成的信号肽,68个氨基酸残基组成的成熟肽。成熟肽原核表达产物为不可溶的包涵体。经变性、复性后纯化的r Na Ku I1无抗凝血活性。在100倍摩尔浓度比下,r Na Ku I1对人纤溶酶(5 nmol/L)、人胰蛋白酶(1 nmol/L)和猪胰蛋白酶(5 nmol/L)活性的抑制率近100%,对牛α-胰糜蛋白酶(1 nmol/L)和人中性粒细胞弹性蛋白酶(5 nmol/L)活性的抑制率分别约31.45%和25.18%,对人组织蛋白酶G、蛋白酶3和猪胰弹性蛋白酶均无抑制作用。r Na Ku I1抑制人胰蛋白酶及纤溶酶的抑制常数(ki)分别为(21.17±7.22)和(21.72±3.95)nmol/L。结论成功分离获得Na Ku I1全长c DNA序列,其原核表达产物r Na Ku I1具有较强抑制胰蛋白酶和纤溶酶活性的特点。
