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

Expression and purification of recombinant human serum albumin from selectively terminable transgenic rice first-author corresp ‡Corresponding Author ‡Corresponding Author †E-mail:zcshen@zju.edu.cn †E-mail:zcshen@zju.edu.cn zcshen@zju.edu.cn State Key Laboratory of Rice Biology and Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China State Key Laboratory of Rice Biology and Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China 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. Recombinant human serum albumin (rHSA) Selectively terminable transgenic rice Purification Introduction 1 Human serum albumin (HSA) is the most widely used human plasma protein. It contains a single unglycosylated polypeptide chain of 585 amino acids (66.5 kDa) in its globular structure (Huang et al., 2005 2005 ; Belew et al., 2008 2008 ). HSA is widely used to treat severe hypoproteinemia and hyperbilirubinemia, post-surgery and post-traumatic shock, and hepatocirrhosis (Hastings and Wolf, 1992 1992 ; Mendez et al., 2005 2005 ). Aside from its major use as a blood volume expander, HSA is also frequently used in biochemical applications, such as the formulation of protein therapeutics, cell culture media, drug delivery, cryopreservation, in vivo diagnostics, vaccine formulation and manufacturing, and infertility treatments (Hammitt et al., 1991 1991 ; Marth and Kleinhappl, 2001 2001 ; Langer et al., 2003 2003 ; Cai et al., 2006 2006 ; Subramanian et al., 2007 2007 ; Kratz, 2008 2008 ; Tsuchida et al., 2009 2009 ). Traditionally, the plasma HSA (pHSA) has been obtained from human blood, which is limited in supply and may carry a risk of viral infections, e.g., human immunodeficiency virus (HIV) and hepatitis (Erstad, 1996 1996 ). As the commercial HSA market increases continuously worldwide, recombinant HSA (rHSA) offers a highly attractive way to meet current and future demand. The physicochemical and immunochemical properties of rHSA have been analyzed previously (Ohtani et al., 1998 1998 ; Bosse et al., 2005 2005 ; He et al., 2011 2011 ), and found to be comparable in safety and tolerability to those of its natural counterpart, pHSA. Aside from the traditional yeast expression system (Chuang and Otagiri, 2007 2007 ), various genetically modified plants have been explored to express heterogeneous HSA, including potato (Farran et al., 2002 2002 ), tobacco (Dong et al., 2012 2012 ), and rice (Huang et al., 2005 2005 ). Among the different plant platforms, transgenic rice seed shows great potential for HSA expression because of its low-cost production feasibility (Huang et al., 2007 2007 ; He et al., 2011 2011 ). However, the major concern about using rice to produce rHSA is unintentional spreading of the transgene into the food chain. Although physical containment measures (isolation zones, buffer zones, fencing, etc.) have been taken to contain transgenes, incidents of transgene escape have still occurred (Fox, 2003 2003 ; Vermij, 2006 2006 ; Ledford, 2007 2007 ). No method has yet been reported that involves using chemical traits to prevent transgenes from spreading into regular rice, and which may be applicable to an rHSA bioreactor system. Therefore, a simple and reliable containment method is highly desirable from a biosafety point of view. Since purifying recombinant proteins from plant biomass may represent up to more than 80% of the final product costs (Kusnadi et al., 1997 1997 ), a simplified downstream scheme is required urgently to refine rHSA for commercially acceptable production. Based on our previous work (Lin et al., 2008 2008 ), we have developed a transgenic rice expression system for rHSA with a novel built-in transgene containment technology. An rHSA expression cassette is coupled with a glyphosate resistance gene and an RNA interference (RNAi) cassette rendering bentazon susceptivity. In this study, we report the creation of a transgenic line, which stably expresses high levels of rHSA in seeds and can be selectively terminated by bentazon to contain its spread. We also describe a simple and optimized scheme for rHSA purification. Materials and methods 1 Vector construction 2 The HSA sequence from Homo sapiens (gb:CAA01491) was optimized with a rice codon bias and synthesized by Shanghai Sangon Co., Ltd., China. A corn phosphoenolpyruvate carboxylase (PEPC) terminator was added after the stop codon. An Xba I site was introduced at the 5′-end and a Kpn I site was added to the 3′-end of the synthetic gene. This synthetic gene was linked to a DNA fragment consisting of the rice glutelin Gt1 promoter and its signal peptide to target the rHSA into the rice seeds. The Gt1 promoter and the synthetic HSA gene including the terminator were digested with Hin dIII/ Xba I and Xba I/ Kpn I, respectively, and ligated in a three-way ligation into the pCAMBIA1300 vector backbone (CAMBIA, Australia) pre-digested with Hin dIII and Kpn I. The resulting vector was named p1300-HSA. The fragment G6-P450-RNAi, consisting of glyphosate tolerance 5-enolpyruvylshikimate-3-phosphate synthase ( EPSPS ) gene G6 (gb:EU169459) directed by the Zea mays polyubiquitin-1 promoter (ZmUbi) and the reverse repeat sequence for RNAi against CYP81A6 (gb:DQ341412) (Pan et al., 2006 2006 ) directed by the cauliflower mosaic virus 35S promoter (CaMV35S), was released from the plasmid pG6-450i by Kpn I and Xho I digestion as described previously (Lin et al., 2008 2008 ). This G6-P450-RNAi cassette was linked to the plasmid p1300-HSA pre-digested with Kpn I and Xho I. The resulting binary vector for rice transformation, named pCAMBIA1300-HSA-G6-P450-RNAi, includes the HSA expression, glyphosate resistance, and RNAi cassettes (Fig. 1 1 ). 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 Rice transformation 2 The T-DNA transformation construct pCAMBIA1300-HSA-G6-P450-RNAi was introduced into Agrobacterium tumefaciens (LBA4404) using an electroporator (Eppendorf, Germany) according to the manufacturer’s instructions. A local rice cultivar “Xiushui-110” ( Oryza sativa L. ssp. japonica ) was transformed using the method reported previously with minor modifications (Hiei et al., 1994 1994 ). Glyphosate (Sigma, USA) with a final concentration of 2 mmol/L was used as the selection agent. Spraying of herbicides 2 Plants of transgenic rice line HSA-84 and untransformed rice were grown in solution in the greenhouse. Herbicide spray tests were carried out when the height of the rice seedlings reached about 20 cm. The plants were all sprayed with either bentazon or glyphosate using a handheld sprayer. For the glyphosate tolerance test, 41% propylamine amine salt of glyphosate (Roundup®, Monsanto, USA) was diluted to a final concentration of 0.2% for spraying. For the bentazon susceptibility assay, a 48% bentazon solution (Basagran®, BASF, Germany) was sprayed at a concentration of 2 000 mg/L. Protein analysis and quantification 2 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analyses were performed using a Mini-Protean® Tetra Cell Electrophoresis System (Bio-Rad, USA) with 12% gels. The protein concentrations were measured using the Bradford ( 1976 1976 )’s method with 99% purity commercial pHSA (ProSpec, USA) as the standard. The absorbance of the samples at 595 nm was recorded with a VersaMax ELISA Microplate Reader (Molecular Devices, USA). The quantification of rHSA was determined using the ELISA Human Albumin Quantification Kit (Bethyl Laboratories, USA) based on the manufacturer’s instructions. Isolation and purification of rHSA from transgenic rice seeds 2 The powder from transgenic rice seeds was milled in 25 mmol/L phosphate buffer (PBS; pH 8.0) at a ratio of 1:5 (w/v) for 2 h to obtain a crude extract. After the addition of 10 mmol/L sodium caprylate, the crude extract was immersed in a thermostatic water bath for 30 min at 68 °C. The mixture was precipitated at pH 4.5 for 4 h at room temperature. The supernatant was adjusted to pH 8.0 before being loaded onto an ANX Sepharose Fast Flow (ANX FF; GE Healthcare, USA) column that had been equilibrated with 25 mmol/L PBS (pH 8.0) prior to sample application. The adsorbed rHSA was eluted with 25 mmol/L PBS containing 150 mmol/L NaCl (pH 8.0). The eluted peak containing the target protein was further applied to a Butyl Sepharose High Performance (Butyl HP; GE Healthcare) column equilibrated with buffer containing 25 mmol/L PBS and 1.2 mol/L ammonium sulfate, at pH 7.0. The binding targeted protein was finally eluted with buffer containing 25 mmol/L PBS and 0.5 mol/L ammonium sulfate, at pH 7.0. The rHSA fraction was desalted and concentrated by ultrafiltration using an Amicon Ultra-15 tube (Millipore, Germany). All chromatographic experiments were performed at room temperature using an AKTA explorer 100 (GE Healthcare) fast protein liquid chromatography (FPLC) system. A linear flow rate of 150 cm/h was used throughout. The final purity of rHSA was determined using a Fast Silver Stain Kit (Beyotime, China). Molecular mass determination, N-terminus sequencing, and circular dichroism (CD) spectrum determination 2 To further characterize rHSA extracted from rice grain, the purified protein was subjected to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis using a Bruker AutoflexII mass spectrometer (Bruker Daltonic, USA) from Shanghai Applied Protein Technology Co., Ltd., China. The mass spectrometry data of samples were calculated using Mascot software (Matrix Science, UK) to identify proteins in the Swiss-Prot database. For N-terminal amino acid residue determination, total soluble protein extracted from rice seeds was separated by 12% SDS-PAGE followed by electroblotting onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, USA). The membrane was stained with coomassie brilliant blue R-250, and the band corresponding to rHSA was excised for Edman degradation. N-terminal sequencing was carried out using an ABI491A (Applied Biosystems, USA) sequencer from Shanghai Applied Protein Technology Co., Ltd., China. The CD spectra for rHSA and pHSA were recorded on a JASCO J-815 automatic spectropolarimeter by the Analysis and Measurement Center of Zhejiang University of Technology, China. The concentration of protein was 1 mg/ml in 50 mmol/L PBS (pH 7.4). Data were measured in the range of 190–390 nm at a scanning speed of 50 nm/min. Results 1 Creation of transgenic rice stably expressing HSA 2 A total of 209 independent transformed T0 events were generated and grown in the field to harvest seeds. The total soluble protein extracted from T1 rice seeds was analyzed by SDS-PAGE for selecting transgenic lines with high rHSA expression level. A line named HSA-84 was found to be one of the highest expressing lines (Fig. 2 2 ). The transgenic rice seeds of HSA-84 displayed an opaque phenotype compared with non-transgenic control seeds. Due to the obvious visual difference, it was easy to distinguish the transgenic seeds from the regular seeds. We found that the transgenic rice seeds weighed an average of (20.4±1.2) g/1 000 grains compared to (23.1±1.6) g/1 000 grains for the conventional rice of the same cultivar, suggesting nearly a 10% yield penalty in the HSA-84 line. The germination rate of the transgenic rice was 92%, which was very close to the 94% of the conventional rice. The terminable transgenic rice showed no visible difference in growth and development compared to the non-transgenic control. rHSA was estimated to account for 0.72% of the total dry weight of the dehusked rice seeds. No significant difference was found among the T0–T3 seeds in rHSA expression level, indicating that the rHSA was stably expressed in different generations. Since the genetic cassettes for HSA expression, RNAi of the bentazon detoxifying enzyme, and glyphosate tolerance were all in a single T-DNA fragment, the chance of separation among these three cassettes was extremely low. 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) Selective termination of HSA-84 plants by herbicides 2 The T3 plants of HSA-84 were tested to determine their susceptibility to bentazon and glyphosate in the greenhouse. We found that one spray of bentazon at 2 000 mg/L killed 100% of plants of HSA-84, but none of the regular non-transgenic rice, within 10 d (Fig. 3a 3a ). Thus, the regular dose of 2 000 mg/L used for normal rice weed control is enough to kill the transgenic rice HSA-84. In contrast, the spraying of 20 mmol/L glyphosate killed all of the conventional rice plants in 10 d but did not affect any transgenic plants, as expected (Fig. 3b 3b ). These tests clearly demonstrated that the transgenic event HSA-84 was highly sensitive to bentazon but tolerant to glyphosate, while the conventional rice plants showed the opposite responses. Thus, the termination of the transgenic rice plants could be highly feasible. Moreover, similar results were observed after further tests on T4 and T5 plants, suggesting that the sensitivity to the two herbicides was stably inherited in transgenic line HSA-84. However, more generations of transgenic plants still need to be monitored as the long-term heritability of the RNAi has not yet been well studied. 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 rHSA purification scheme 2 A purification scheme comprising crude extract pretreatment and two chromatography steps was developed in this study (Fig. 4 4 ). Since HSA has a high thermal tolerance (Sumi et al., 1999 1999 ; Belew et al., 2008 2008 ) but high protease-sensitivity (Fernández-San Millán et al., 2003 2003 ), thermal treatment of crude protein extracts was performed to inactivate proteases and denature some rice seed storage proteins. Initially, we attempted to use Capto MMC (Belew et al., 2008 2008 ) to capture rHSA. Unexpectedly, we found precipitation at pH 4.5 could significantly remove a large quantity of other proteins (Table 1 1 ). Based on this finding, a precipitation procedure, rather than a chromatography step, was used for the initial step in the purification scheme. In the presence of 10 mmol/L sodium caprylate, rHSA was protected from degradation during the heating and precipitation processes with a recovery rate of 90.5% (Table 1 1 ). Accordingly, this stabilizer should be dissolved into the crude extract prior to heat treatment. Note that crude extracts with high turbidity could be clarified after heating and precipitating, and thus, centrifugation is not necessary before the chromatography step. Recovery=(rHSA weight in each step/rHSA weight in initial extraction)×100%; Purity=(rHSA weight/TSP weight in the same step)×100%, where TSP is total soluble protein Purity of rHSA was determined by SDS-PAGE (silver staining) Values are expressed as mean±standard deviation (SD), n =3 Purification step rHSA weight (mg) Purity (%) Recovery (%) Initial extraction 288±19 29.3±3.2 100 Heat treatment & pH adjustment 259±35 59.6±1.9 90.5±3.2 ANX FF 224±24 87.7±2.3 85.8±2.6 Butyl HP 180±36 > 99 * * 80.4±3.9 Final preparation 179±6 > 99 * * 62.4±3.7 A capture purification step was started with ANX FF, which proved to be effective for rHSA concentration and partial purification. This step enriched rHSA to 85.8% (Table 1 1 ), whereas the major low molecular weight proteins in the supernatant were removed in the flow through fraction (Fig. 5 5 ). As a polishing step, the Butyl HP column was used to facilitate the removal of the residual non-HSA proteins. The rHSA eluted from Butyl HP column appeared as a single band of pure protein in SDS-PAGE (Fig. 5 5 ). 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 The total recovery of rHSA from triple replications was (62.4±3.7)% (Table 1 1 ), indicating that the complete purification process is reproducible. SDS-PAGE silver-staining analysis demonstrated that the final purity of rHSA was comparable to that of a pHSA ( > 99%) control. Together, these results suggest that the rHSA was successfully purified by the two steps of chromatographic purification. Characterization of the purified rHSA 2 Mass spectrometry analysis, N-terminus sequencing, and determination of the CD spectrum were performed to confirm the similarity of rHSA to the natural protein. The m / z (mass/charge) value showed that the rHSA had an equivalent molecular weight of 66 441 Da (Fig. 6 6 ), which is very close to the 66 531 Da of its natural counterpart from the UniProtKB database. Ten N-terminal amino acid residues were determined as follows: DAHKSEVAHR. This result revealed that the cleavage of the signal peptide sequence was correct and that the mature rHSA was identical to the pHSA in the primary structure. The correct secondary structure is necessary for the functional activity of rHSA. The CD spectrum analysis further confirmed that the recombinant protein had the same conformation as the natural analogue (Fig. 7 7 ). Discussion 1 While the technology for producing rHSA using transgenic rice has been well developed, the issue of a transgene containment strategy has not been addressed for transgenic rice for molecular farming. This is a major concern for large scale planting of such transgenic rice in open fields. We have developed a transgenic line for high expression of rHSA with a built-in spread control technology. Such transgenic rice plants could be killed by bentazon during regular weed control if they spread accidently into regular rice. The technology could be used as a preventive scheme to exclude any possibly contaminated transgenic rice expression of rHSA. When a certain area of rice is suspected of being contaminated, we can ensure removal of any rHSA contaminant simply by using bentazon as the herbicide for weed control. The built-in containment strategy illustrated in this study makes the detection and selective termination of the transgenic plants inexpensive and convenient. Rice-derived rHSA does not require sophisticated equipment, unlike traditional yeast fermentation, and could be scaled up simply by planting a larger acreage of transgenic rice. Rice seeds also offer a low hydrolytic condition for storage of rHSA, which can overcome the limitation of protein stability within the microbial expression system. The concentration of rHSA in T3 transgenic rice seeds was shown to account for 0.72% of dry weight of the dehusked rice seeds in our assays, which is much higher than the estimated cost-effective threshold (0.1 g/kg) for commercial production of rHSA in plants (Farran et al., 2002 2002 ). Another object of this study was to develop a good manufacturing practice (GMP)-compliant scheme for isolating rHSA of high purity. The total recovery of our purification process was 62.4% and the final product reached 99% purity, which is likely to be pure enough for use as a cell culture component. The chromatography combination (ANX FF and Butyl HP) designed in this study significantly shortened the purification period and reduced the operational cost. We believe that the simple purification protocol could be further optimized for pilot- or production-scale operations. We have proved that the rHSA produced by the transgenic rice reported here is identical to native protein from blood in terms of its N-terminal amino acid sequence, molecular mass as measured by MALDI-TOF, and secondary structure as shown by its CD spectrum. Previous research has shown that rHSA is identical to native protein in biological activity (Kobayashi, 2006 2006 ). Therefore, we expect that the rHSA protein produced by the transgenic rice here is also biologically identical to its native protein. In conclusion, we have developed a commercially feasible transgenic rice expression system for rHSA with a built-in containment technology. Unlike non-transgenic rice, such transgenic rice with high glyphosate resistance is extremely sensitive to bentazon, and thus can be selectively killed by bentazon. We also designed a simple purification scheme with high efficiency and high yield, which can purify the rHSA to 99% purity. References M Belew , M Yan , W Zhang , K Caldwell . Purification of recombinant human serum albumin (rHSA) produced by genetically modified Pichia pastoris . Separ Sci Technol , 2008 . 43 ( 11-12 ): 3134 - 3153 . DOI: 10.1080/01496390802221857 http://doi.org/10.1080/01496390802221857 . Purification of recombinant human serum albumin (rHSA) produced by genetically modified Pichia pastoris D Bosse , M Praus , P Kiessling , L Nyman , C Andresen , J Waters , F Schindel . Phase I comparability of recombinant human albumin and human serum albumin . J Clin Pharmacol , 2005 . 45 ( 1 ): 57 - 67 . 15601806 DOI: 10.1177/0091270004269646 http://doi.org/10.1177/0091270004269646 . Phase I comparability of recombinant human albumin and human serum albumin MM Bradford . A rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding . Anal Biochem , 1976 . 72 ( 1-2 ): 248 - 254 . 942051 DOI: 10.1016/0003-2697(76)90527-3 http://doi.org/10.1016/0003-2697(76)90527-3 . A rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding C Cai , K Zhou , Y Wu , L Wu . Enhanced liver targeting of 5-fluorouracil using galactosylated human serum albumin as a carrier molecule . J Drug Target , 2006 . 14 ( 2 ): 55 - 61 . 16608732 DOI: 10.1080/10611860600613324 http://doi.org/10.1080/10611860600613324 . Enhanced liver targeting of 5-fluorouracil using galactosylated human serum albumin as a carrier molecule VTG Chuang , M Otagiri . Recombinant human serum albumin . Drugs Today , 2007 . 43 ( 8 ): 547 - 561 . 17925886 DOI: 10.1358/dot.2007.43.8.1067343 http://doi.org/10.1358/dot.2007.43.8.1067343 . Recombinant human serum albumin Y Dong , F Zhang , Z Wang , L Du , A Hao , B Jiang , M Tian , Q Li , Q Jia , S Wang , 等 . Extraction and purification of recombinant human serum albumin from Pichia pastoris broths using aqueous two-phase system combined with hydrophobic interaction chromatography . J Chromatogr A , 2012 . 1245 143 - 149 . 22658659 DOI: 10.1016/j.chroma.2012.05.041 http://doi.org/10.1016/j.chroma.2012.05.041 . Extraction and purification of recombinant human serum albumin from Pichia pastoris broths using aqueous two-phase system combined with hydrophobic interaction chromatography BL Erstad . Viral infectivity of albumin and plasma protein fraction . Pharmacotherapy , 1996 . 16 ( 6 ): 996 - 1001 . 8947970 . Viral infectivity of albumin and plasma protein fraction I Farran , J Sánchez-Serrano , J Medina , J Prieto , A Mingo-Castel . Targeted expression of human serum albumin to potato tubers . Transgenic Res , 2002 . 11 ( 4 ): 337 - 346 . 12212837 DOI: 10.1023/A:1016356510770 http://doi.org/10.1023/A:1016356510770 . Targeted expression of human serum albumin to potato tubers A Fernández-San Millán , A Mingo-Castel , M Miller , H Daniell . A chloroplast transgenic approach to hyper-express and purify human serum albumin, a protein highly susceptible to proteolytic degradation . Plant Biotechnol J , 2003 . 1 ( 2 ): 71 - 79 . 17147744 DOI: 10.1046/j.1467-7652.2003.00008.x http://doi.org/10.1046/j.1467-7652.2003.00008.x . A chloroplast transgenic approach to hyper-express and purify human serum albumin, a protein highly susceptible to proteolytic degradation JL Fox . Puzzling industry response to ProdiGene fiasco . Nat Biotechnol , 2003 . 21 ( 1 ): 3 - 4 . 12511893 DOI: 10.1038/nbt0103-3b http://doi.org/10.1038/nbt0103-3b . Puzzling industry response to ProdiGene fiasco D Hammitt , D Walker , C Syrop , T Miller , M Bennett . Treatment of severe male-factor infertility with high concentrations of motile sperm by microinsemination in embryo cryopreservation straws . J In Vitro Fert Embryo Transf , 1991 . 8 ( 2 ): 101 - 110 . 2061678 DOI: 10.1007/BF01138663 http://doi.org/10.1007/BF01138663 . Treatment of severe male-factor infertility with high concentrations of motile sperm by microinsemination in embryo cryopreservation straws GE Hastings , PG Wolf . The therapeutic use of albumin . Arch Fam Med , 1992 . 1 ( 2 ): 281 - 287 . 1341604 DOI: 10.1001/archfami.1.2.281 http://doi.org/10.1001/archfami.1.2.281 . The therapeutic use of albumin Y He , T Ning , T Xie , Q Qiu , L Zhang , Y Sun , D Jiang , K Fu , F Yin , W Zhang , 等 . Large-scale production of functional human serum albumin from transgenic rice seeds . PNAS , 2011 . 108 ( 47 ): 19078 - 19083 . 22042856 DOI: 10.1073/pnas.1109736108 http://doi.org/10.1073/pnas.1109736108 . Large-scale production of functional human serum albumin from transgenic rice seeds Y Hiei , S Ohta , T Komari , T Kumashiro . Efficient transformation of rice ( Oryza sativa L.) mediated by agrobacterium and sequence analysis of the boundaries of the T-DNA . Plant J , 1994 . 6 ( 2 ): 271 - 282 . 7920717 DOI: 10.1046/j.1365-313X.1994.6020271.x http://doi.org/10.1046/j.1365-313X.1994.6020271.x . Efficient transformation of rice (Oryza sativa L.) mediated by agrobacterium and sequence analysis of the boundaries of the T-DNA LF Huang , YK Liu , CA Lu , SL Hsieh , SM Yu . Production of human serum albumin by sugar starvation induced promoter and rice cell culture . Transgenic Res , 2005 . 14 ( 5 ): 569 - 581 . 16245148 DOI: 10.1007/s11248-004-6481-5 http://doi.org/10.1007/s11248-004-6481-5 . Production of human serum albumin by sugar starvation induced promoter and rice cell culture N Huang , RL Rodriquez , FE Hagie , DM Stalker . Expression of Human Serum Albumin (HSA) in Monocot Seeds . US 07304208 . 2007 . Expression of Human Serum Albumin (HSA) in Monocot Seeds K Kobayashi . Summary of recombinant human serum albumin development . Biologicals , 2006 . 34 ( 1 ): 55 - 59 . 16464610 DOI: 10.1016/j.biologicals.2005.08.021 http://doi.org/10.1016/j.biologicals.2005.08.021 . Summary of recombinant human serum albumin development F Kratz . Albumin as a drug carrier: design of prodrugs, drug conjugates and nanoparticles . J Control Release , 2008 . 132 ( 3 ): 171 - 183 . 18582981 DOI: 10.1016/j.jconrel.2008.05.010 http://doi.org/10.1016/j.jconrel.2008.05.010 . Albumin as a drug carrier: design of prodrugs, drug conjugates and nanoparticles AR Kusnadi , ZL Nikolov , JA Howard . Production of recombinant proteins in transgenic plants: practical considerations . Biotechnol Bioeng , 1997 . 56 ( 5 ): 473 - 484 . 18642268 DOI: 10.1002/(SICI)1097-0290(19971205)56:5<473::AID-BIT1>3.3.CO;2-4 http://doi.org/10.1002/(SICI)1097-0290(19971205)56:5<473::AID-BIT1>3.3.CO;2-4 . Production of recombinant proteins in transgenic plants: practical considerations K Langer , S Balthasar , V Vogel , N Dinauer , H von Briesen , D Schubert . Optimization of the preparation process for human serum albumin (HSA) nanoparticles . Int J Pharm , 2003 . 257 ( 1-2 ): 169 - 180 . 12711172 DOI: 10.1016/S0378-5173(03)00134-0 http://doi.org/10.1016/S0378-5173(03)00134-0 . Optimization of the preparation process for human serum albumin (HSA) nanoparticles H Ledford . Out of bounds . Nature , 2007 . 445 ( 7124 ): 132 - 133 . 17215811 DOI: 10.1038/445132a http://doi.org/10.1038/445132a . Out of bounds CY Lin , J Fang , XL Xu , T Zhao , JA Cheng , JM Tu , GY Ye , ZC Shen . A built-in strategy for containment of transgenic plants: creation of selectively terminable transgenic rice . PloS One , 2008 . 3 ( 3 ): e1818 18350155 DOI: 10.1371/journal.pone.0001818 http://doi.org/10.1371/journal.pone.0001818 . A built-in strategy for containment of transgenic plants: creation of selectively terminable transgenic rice E Marth , B Kleinhappl . Albumin is a necessary stabilizer of TBE-vaccine to avoid fever in children after vaccination . Vaccine , 2001 . 20 ( 3-4 ): 532 - 537 . 11672919 DOI: 10.1016/S0264-410X(01)00329-2 http://doi.org/10.1016/S0264-410X(01)00329-2 . Albumin is a necessary stabilizer of TBE-vaccine to avoid fever in children after vaccination CM Mendez , CJ Mcclain , LS Marsano . Albumin therapy in clinical practice . Nutr Clin Pract , 2005 . 20 ( 3 ): 314 - 320 . 16207669 DOI: 10.1177/0115426505020003314 http://doi.org/10.1177/0115426505020003314 . Albumin therapy in clinical practice W Ohtani , Y Nawa , K Takeshima , H Kamuro , K Kobayashi , T Ohmura . Physicochemical and immunochemical properties of recombinant human serum albumin from Pichia pastoris . Anal Biochem , 1998 . 256 ( 1 ): 56 - 62 . 9466797 DOI: 10.1006/abio.1997.2480 http://doi.org/10.1006/abio.1997.2480 . Physicochemical and immunochemical properties of recombinant human serum albumin from Pichia pastoris G Pan , XY Zhang , KD Liu , JW Zhang , XZ Wu , J Zhu , JM Tu . Map-based cloning of a novel rice cytochrome P450 gene CYP81A6 that confers resistance to two different classes of herbicides . Plant Mol Biol , 2006 . 61 ( 6 ): 933 - 943 . 16927205 DOI: 10.1007/s11103-006-0058-z http://doi.org/10.1007/s11103-006-0058-z . Map-based cloning of a novel rice cytochrome P450 gene CYP81A6 that confers resistance to two different classes of herbicides GM Subramanian , M Fiscella , A Lamouse-Smith , S Zeuzem , JG Mchutchison . Albinterferon [α]-2b: a genetic fusion protein for the treatment of chronic hepatitis C . Nat Biotechnol , 2007 . 25 ( 12 ): 1411 - 1419 . 18066038 DOI: 10.1038/nbt1364 http://doi.org/10.1038/nbt1364 . Albinterferon [α]-2b: a genetic fusion protein for the treatment of chronic hepatitis C A Sumi , K Okuyama , K Kobayashi , W Ohtani , T Ohmura , K Yokoyama . Purification of recombinant human serum albumin efficient purification using streamline . Bioseparation , 1999 . 8 ( 1/5 ): 195 - 200 . 10734571 DOI: 10.1023/A:1008081314112 http://doi.org/10.1023/A:1008081314112 . Purification of recombinant human serum albumin efficient purification using streamline E Tsuchida , K Sou , A Nakagawa , H Sakai , T Komatsu , K Kobayashi . Artificial oxygen carriers, hemoglobin vesicles and albumin-hemes, based on bioconjugate chemistry . Bioconjug Chem , 2009 . 20 ( 8 ): 1419 - 1440 . 19206516 DOI: 10.1021/bc800431d http://doi.org/10.1021/bc800431d . Artificial oxygen carriers, hemoglobin vesicles and albumin-hemes, based on bioconjugate chemistry P Vermij . Liberty link rice raises specter of tightened regulations . Nat Biotechnol , 2006 . 24 ( 11 ): 1301 - 1302 . 17093456 DOI: 10.1038/nbt1106-1301 http://doi.org/10.1038/nbt1106-1301 . Liberty link rice raises specter of tightened regulations Compliance with ethics guidelines Qing ZHANG, Hui YU, Feng-zhen ZHANG, and Zhi-cheng SHEN declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1300090 Project (No. 2011ZX08010-003) supported by the Ministry of Agriculture of China 2013-03-26 2013-09-22 2013-06-06 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2013 2013 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 10 14