Papers by SAURABH AGARWAL
Transgenic research, 2008
Transgenic plants offer promising alternative for large scale, sustainable production of safe, fu... more 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 a-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 0 and 3 0 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) sporam-ineA 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 0 while an endoplasmic reticulum retention signal (KDEL) was incorporated at 3 0 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 a-1-antitrypsin in transgenic tomato plants.
Plant Science, 2012
The response of protein accumulation site on yield, biological activity and in planta stability o... more The response of protein accumulation site on yield, biological activity and in planta stability of therapeutic recombinant human ␣ 1 -proteinase inhibitor (␣ 1 -PI) was analyzed via targeting to different subcellular locations, like endoplasmic reticulum (ER), apoplast, vacuole and cytosol in leaves of transgenic tomato plants. In situ localization of the recombinant ␣ 1 -PI protein in transgenic plant cells was monitored by immunohistochemical staining. Maximum accumulation of recombinant ␣ 1 -PI in T 0 and T 1 transgenic tomato plants was achieved from 1.5 to 3.2% of total soluble protein (TSP) by retention in ER lumen, followed by vacuole and apoplast, whereas cytosolic targeting resulted into degradation of the protein.
Journal of Biotechnology, 2010
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Papers by SAURABH AGARWAL