Chloroplast expression of His-tagged GUS-fusions: a general strategy to overproduce and purify foreign proteins using transplastomic plants as bioreactors

被引:100
作者
Leelavathi, S [1 ]
Reddy, VS [1 ]
机构
[1] Int Ctr Genet Engn & Biotechnol, New Delhi 110067, India
关键词
affinity chromatography; chloroplast expression; GUS-fusion; His-tag; interferon gamma; TRANSGENIC TOBACCO PLANTS; HUMAN INTERFERON-GAMMA; BACILLUS-THURINGIENSIS; PLASTID TRANSFORMATION; ANTIMICROBIAL PEPTIDE; BIOPHARMACEUTICALS; RESISTANCE; GENOME; OVEREXPRESSION; GLYCOSYLATION;
D O I
10.1023/A:1022114427971
中图分类号
S3 [农学(农艺学)];
学科分类号
0901 ;
摘要
High level expression and efficient recovery of recombinant protein are two main critical factors that determine the use of transgenic plants as natural bioreactors to produce foreign proteins for industrial applications. We demonstrate here the potential of a new strategy involving chloroplast transformation, GUS-fusions and affinity-tag based chromatography to overexpress and purify a human therapeutic protein, interferon gamma (IFN-g) in tobacco plants. Our results show that IFN-g accumulation reaches up to 6% of total soluble protein when expressed as a GUS-fusion protein in tobacco chloroplasts. Addition of His-tag simplified the downstream process and the recombinant protein yields were considerably high (similar to360 mug/g fresh leaf tissue). Further we demonstrate the use of GUS-fusions to identify recombinant protein containing fractions very rapidly (< 5 minutes) through simple GUS assay, an important consideration for those proteins that are highly labile during lengthy and harsh downstream processing conditions. The chloroplast-produced IFN-g is biologically as active as the same protein obtained through E. coli expression without any involvement of refolding procedure. Our results demonstrate that the new strategy has tremendous potential for large scale production of proteins from heterologous source, independent of their physio-chemical and biological properties, using plants as 'natural bioreactors'.
引用
收藏
页码:49 / 58
页数:10
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