Improved expression and purification of recombinant human serum albumin from transgenic tobacco suspension culture

► A cowpea mosaic virus vector was used to express human serum albumin in plant cells. ► Recombinant human serum albumin (rHSA) was highly produced in tobacco BY-2 cells. ► Cultivation of BY-2 cells in pH 8.0 medium improved the yield of rHSA two-fold. ► A simple purification scheme was developed to...

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Bibliographic Details
Published in:Journal of biotechnology 2011-09, Vol.155 (2), p.164-172
Main Authors: Sun, Qiao-Yang, Ding, Ling-Wen, Lomonossoff, George P., Sun, Yong-Bing, Luo, Ming, Li, Chao-Qiong, Jiang, Liwen, Xu, Zeng-Fu
Format: Article
Language:English
Subjects:
Biological and medical sciences
Biotechnology
Biotechnology - methods
Blotting, Western
BY-2 cells
cell culture
Cell Culture Techniques - methods
Cells, Cultured
Comovirus
Cowpea mosaic virus
Culture
culture media
desorption
Electrophoresis, Polyacrylamide Gel
Fundamental and applied biological sciences. Psychology
Genetic Vectors
Human
human serum albumin
Humans
Mass Spectrometry
matrix-assisted laser desorption-ionization mass spectrometry
Nicotiana - metabolism
pEAQ
Plant suspension culture
Plants (organisms)
Plants, Genetically Modified - metabolism
protein synthesis
Proteins
proteolysis
Recombinant
Recombinant HSA
Recombinant Proteins - genetics
Recombinant Proteins - isolation & purification
Recombinant Proteins - metabolism
sequence analysis
Serum albumin
Serum Albumin - genetics
Serum Albumin - isolation & purification
Serum Albumin - metabolism
Signal peptide
Tobacco
Transgenic
transgenic plants
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Summary:► A cowpea mosaic virus vector was used to express human serum albumin in plant cells. ► Recombinant human serum albumin (rHSA) was highly produced in tobacco BY-2 cells. ► Cultivation of BY-2 cells in pH 8.0 medium improved the yield of rHSA two-fold. ► A simple purification scheme was developed to purify the rHSA from culture medium. ► Detailed characterization showed pant cell-derived rHSA was identical to natural HSA. Most human serum albumin (HSA) for medical applications is derived from human plasma due to the lack of suitable heterologous expression systems for recombinant HSA (rHSA). To determine whether plant cell cultures could provide an alternative source, we employed the hyper-translatable cowpea mosaic virus protein expression system (CPMV- HT) to stably express rHSA in tobacco Bright Yellow-2 (BY-2) cells. rHSA was stably produced with yield up to 11.88 μg/ml in the culture medium, accounting for 0.7% of total soluble protein, in a 25-ml flask. Cultivation of transgenic cells in modified Murashige and Skoog medium with a pH of 8.0 improved the yield of rHSA two-fold, which may be the result of reduced proteolytic activity in the modified medium. A simple purification scheme was developed to purify the rHSA from culture medium, resulting in a recovery of 48.41% of the secreted rHSA. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and N-terminal sequence analysis of the purified rHSA revealed that plant cell-derived rHSA is identical to that of the plasma-derived HSA. Our results show that the CPMV- HT system, which was originally developed as a transient expression system for use in whole plants, can also be used for high-level expression of rHSA, a protein highly susceptible to proteolysis, in transgenic tobacco cells.
ISSN:0168-1656
1873-4863
DOI:10.1016/j.jbiotec.2011.06.033