title: Expression of modified gene encoding functional human alpha-1-antitrypsin protein in transgenic tomato plants.
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 alpha-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 approximately 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 ( approximately 45 kDa). Our results demonstrate feasibility for high-level expression of biologically active, glycosylated human alpha-1-antitrypsin in transgenic tomato plants.
authors.affiliation: Plant Transgenic Lab, National Botanical Research Institute, PO Box 436, Rana Pratap Marg, Lucknow, 226001, India.
authors.forename: Saurabh
authors.initials: S
authors.lastname: Agarwal
authors.forename: Rahul
authors.initials: R
authors.lastname: Singh
authors.forename: Indraneel
authors.initials: I
authors.lastname: Sanyal
authors.forename: D V
authors.initials: DV
authors.lastname: Amla
mesh_terms: D001483:Base Sequence; D003062:Codon; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D018506:Gene Expression Regulation, Plant; D006031:Glycosylation; D006801:Humans; D018551:Solanum lycopersicum / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D000515:alpha 1-Antitrypsin / Q000235:genetics*
chemical_list: D003062:Codon; D017931:DNA Primers; D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin