Human serum albumin(HSA) is widely utilized for medical purposes and biochemical research.Transgenic rice has proved to be an attractive bioreactor for mass production of recombinant HSA(rHSA).However,transgene spread...Human serum albumin(HSA) is widely utilized for medical purposes and biochemical research.Transgenic rice has proved to be an attractive bioreactor for mass production of recombinant HSA(rHSA).However,transgene spread is a major environmental and food safety concern for transgenic rice expressing proteins of medical value.This study aimed to develop a selectively terminable transgenic rice line expressing HSA in rice seeds,and a simple process for recovery and purification of rHSA for economical manufacture.An HSA expression cassette was inserted into a T-DNA vector encoding an RNA interference(RNAi) cassette suppressing the CYP81A6 gene.This gene detoxifies the herbicide bentazon and is linked to the 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS) cassette which confers glyphosate tolerance.ANX Sepharose Fast Flow(ANX FF) anion exchange chromatography coupled with Butyl Sepharose High Performance(Butyl HP) hydrophobic interaction chromatography was used to purify rHSA.A transgenic rice line,HSA-84,was obtained with stable expression of rHSA of up to 0.72% of the total dry weight of the dehusked rice seeds.This line also demonstrated high sensitivity to bentazon,and thus could be killed selectively by a spray of bentazon.A two-step chromatography purification scheme was established to purify the rHSA from rice seeds to a purity of 99% with a recovery of 62.4%.Results from mass spectrometry and N-terminus sequencing suggested that the purified rHSA was identical to natural plasma-derived HSA.This study provides an alternative strategy for large-scale production of HSA with a built-in transgene safety control mechanism.展开更多
重组蛋白和单克隆抗体等高剂量生物制品的生产,给下游分离纯化技术带来了巨大挑战,膜分离技术已成为确保现代生物制品纯度、安全和效用的基本技术。在基因重组人血白蛋白的纯化过程中,膜分离技术同样发挥了不可替代的作用。实验筛选出...重组蛋白和单克隆抗体等高剂量生物制品的生产,给下游分离纯化技术带来了巨大挑战,膜分离技术已成为确保现代生物制品纯度、安全和效用的基本技术。在基因重组人血白蛋白的纯化过程中,膜分离技术同样发挥了不可替代的作用。实验筛选出适合工业化生产的膜组件UFP-750,并优化了操作工艺。实验结果表明,膜组件UFP-750较其他膜组件,膜过滤滤液质量稳定,产品收率较高,选择的最佳控制参数为跨膜压差为50 k Pa、操作温度为15℃。展开更多
基金Project(No.2011ZX08010-003)supported by the Ministry of Agriculture of China
文摘Human serum albumin(HSA) is widely utilized for medical purposes and biochemical research.Transgenic rice has proved to be an attractive bioreactor for mass production of recombinant HSA(rHSA).However,transgene spread is a major environmental and food safety concern for transgenic rice expressing proteins of medical value.This study aimed to develop a selectively terminable transgenic rice line expressing HSA in rice seeds,and a simple process for recovery and purification of rHSA for economical manufacture.An HSA expression cassette was inserted into a T-DNA vector encoding an RNA interference(RNAi) cassette suppressing the CYP81A6 gene.This gene detoxifies the herbicide bentazon and is linked to the 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS) cassette which confers glyphosate tolerance.ANX Sepharose Fast Flow(ANX FF) anion exchange chromatography coupled with Butyl Sepharose High Performance(Butyl HP) hydrophobic interaction chromatography was used to purify rHSA.A transgenic rice line,HSA-84,was obtained with stable expression of rHSA of up to 0.72% of the total dry weight of the dehusked rice seeds.This line also demonstrated high sensitivity to bentazon,and thus could be killed selectively by a spray of bentazon.A two-step chromatography purification scheme was established to purify the rHSA from rice seeds to a purity of 99% with a recovery of 62.4%.Results from mass spectrometry and N-terminus sequencing suggested that the purified rHSA was identical to natural plasma-derived HSA.This study provides an alternative strategy for large-scale production of HSA with a built-in transgene safety control mechanism.
文摘重组蛋白和单克隆抗体等高剂量生物制品的生产,给下游分离纯化技术带来了巨大挑战,膜分离技术已成为确保现代生物制品纯度、安全和效用的基本技术。在基因重组人血白蛋白的纯化过程中,膜分离技术同样发挥了不可替代的作用。实验筛选出适合工业化生产的膜组件UFP-750,并优化了操作工艺。实验结果表明,膜组件UFP-750较其他膜组件,膜过滤滤液质量稳定,产品收率较高,选择的最佳控制参数为跨膜压差为50 k Pa、操作温度为15℃。
