Differential subcellular targeting of recombinant human α1-proteinase inhibitor influences yield, biological activity and in planta stability of the protein in transgenic tomato plants

The response of protein accumulation site on yield, biological activity and in planta stability of therapeutic recombinant human alpha(1)-proteinase inhibitor (alpha(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 alpha(1)-PI protein in transgenic plant cells was monitored by immunohistochemical staining. Maximum accumulation of recombinant alpha(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. The plant-derived recombinant alpha(1)-PI showed biological activity for elastase inhibition, as monitored by residual porcine pancreatic elastase (PPE) activity assay and band-shift assay. Recombinant alpha(1)-PI was purified from transgenic tomato plants with high yield, homogeneity and biological activity. Purified protein appeared as a single band of similar to 48-50 kDa on SDS-PAGE with pI value ranging between 5.1 and 5.3. Results of mass spectrometry and optical spectroscopy of purified recombinant alpha(1)-PI revealed the structural integrity of the recombinant protein comparable to native serum alpha(1)-PI. Enzymatic deglycosylation and lectin-binding assays with the purified recombinant alpha(1)-PI showed compartment-specific N-glycosylation of the protein targeted to ER, apoplast and vacuole. Conformational studies based on urea-induced denaturation and circular dichroism (CD) spectroscopy revealed relatively lower stability of the recombinant alpha(1)-PI protein, compared to its serum counterpart. Pharmacokinetic evaluation of plant derived recombinant and human plasma-purified alpha(1)-PI in rat, by intravenous route, revealed significantly faster plasma clearance and lower area under curve (AUC) of recombinant protein. Our data suggested significance of protein sorting sequences and feasibility to use transgenic plants for the production of stable, glycosylated and biologically active recombinant alpha(1)-PI for further therapeutic applications. (C) 2012 Elsevier Ireland Ltd. All rights reserved.