摘要
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.
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.
基金
Project(No.2011ZX08010-003)supported by the Ministry of Agriculture of China
