Expression and purification of recombinant human serum albumin from selectively terminable transgenic rice

Abstract

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.

Fig. 1

Diagram of the T-DNA containing the rHSA expression cassette for rice transformation LB and RB: left and right borders of the T-DNA, respectively; Gt1: rice glutelin Gt1 promoter; HSA: human serum albumin; PEPC: corn phosphoenolpyruvate carboxylase terminator; ZmUbi: Zea mays polyubiquitin-1 promoter; G6: 5-enolpyruvylshikimate-3-phosphate synthase isolated from Pseudomonas putida fused with chloroplast transit peptide at the N-terminus; t35S: cauliflower mosaic virus 35S terminator; p35S: cauliflower mosaic virus 35S promoter; P450-RNAi: reverse repeat sequence for RNA interference against CYP81A6

Fig. 2

SDS-PAGE analysis of rHSA in T3 seeds of transgenic rice HSA-84 M: prestained protein ladder; Lane 1: non-transgenic rice seed extract (negative control); Lane 2: seed extract of the T3 transgenic rice HSA-84; Lane 3: pHSA (positive control)

Fig. 3

Susceptibility to bentazon and glyphosate of the transgenic rice HSA-84 The T3 transgenic rice line HSA-84 along with untransformed rice (CK) was cultured in a greenhouse and sprayed with 2 000 mg/L bentazon (a) or 20 mmol/L glyphosate (b). The pictures were taken 10 d after spraying

Fig. 4

Scheme showing the steps involved in the purification of rHSA from the transgenic rice seeds

Fig. 5

SDS-PAGE analysis of the main fractions after different stages of the rHSA purification process Lane 1: crude extract; Lane 2: supernatant after heating and pH adjustment; Lane 3: eluted from ANX FF column; Lane 4: eluted from the Butyl HP column; Lane 5: pHSA

Fig. 6

Molecular weight determination of rHSA by MALDI-TOF

Fig. 7

CD spectra of pHSA (a) and rHSA (b)