NDLI: Expression of modified gene encoding functional human α-1-antitrypsin protein in transgenic tomato plants

Content Provider	Springer Nature Link
Author	Agarwal, Saurabh Singh, Rahul Sanyal, Indraneel Amla, D. V.
Copyright Year	2008
Abstract	Transgenic plants offer promising alternative for large scale, sustainable production of safe, functional, recombinant proteins of therapeutic and industrial importance. Here, we report the expression of biologically active human α-1-antitrypsin in transgenic tomato plants. The 1,182 bp cDNA sequence of human AAT was strategically designed, modified and synthesized to adopt codon usage pattern of dicot plants, elimination of mRNA destabilizing sequences and modifications around 5′ and 3′ flanking regions of the gene to achieve high-level regulated expression in dicot plants. The native signal peptide sequence was substituted with modified signal peptide sequence of tobacco (Nicotiana tabacum) pathogenesis related protein PR1a, sweet potato (Ipomoea batatas) sporamineA and with dicot-preferred native signal peptide sequence of AAT gene. A dicot preferred translation initiation context sequence, 38 bp alfalfa mosaic virus untranslated region were incorporated at 5′ while an endoplasmic reticulum retention signal (KDEL) was incorporated at 3′ end of the gene. The modified gene was synthesized by PCR based method using overlapping oligonucleotides. Tomato plants were genetically engineered by nuclear transformation with Agrobacterium tumefaciens harbouring three different constructs pPAK, pSAK and pNAK having modified AAT gene with different signal peptide sequences under the control of CaMV35S duplicated enhancer promoter. Promising transgenic plants expressing recombinant AAT protein upto 1.55% of total soluble leaf protein has been developed and characterized. Plant-expressed recombinant AAT protein with molecular mass of around ∼50 kDa was biologically active, showing high specific activity and efficient inhibition of elastase activity. The enzymatic deglycosylation established proper glycosylation of the plant-expressed recombinant AAT protein in contrast to unglycosylated rAAT expressed in E. coli (∼45 kDa). Our results demonstrate feasibility for high-level expression of biologically active, glycosylated human α-1-antitrypsin in transgenic tomato plants.
Starting Page	881
Ending Page	896
Page Count	16
File Format	PDF
ISSN	09628819
Journal	Transgenic Research
Volume Number	17
Issue Number	5
e-ISSN	15739368
Language	English
Publisher	Springer Netherlands
Publisher Date	2008-03-05
Publisher Place	Dordrecht
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Human α-1-antitrypsin Codon optimization Modified synthetic gene Heterologous gene expression Transgenic tomato Glycosylation Human Genetics Plant Sciences Animal Genetics and Genomics Plant Genetics & Genomics Molecular Medicine
Content Type	Text
Resource Type	Article
Subject	Genetics Animal Science and Zoology Biotechnology Agronomy and Crop Science

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