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 α1-proteinase inhibitor (α1-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 α1-PI protein in transgenic plant cells was monitored by immunohistochemical staining. Maximum accumulation of recombinant α1-PI in T0 and T1 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 α1-PI showed biological activity for elastase inhibition, as monitored by residual porcine pancreatic elastase (PPE) activity assay and band-shift assay. Recombinant α1-PI was purified from transgenic tomato plants with high yield, homogeneity and biological activity. Purified protein appeared as a single band of ∼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 α1-PI revealed the structural integrity of the recombinant protein comparable to native serum α1-PI. Enzymatic deglycosylation and lectin-binding assays with the purified recombinant α1-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 α1-PI protein, compared to its serum counterpart. Pharmacokinetic evaluation of plant derived recombinant and human plasma-purified α1-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 α1-PI for further therapeutic applications.

► Response of protein accumulation site on yield, stability, functionality and glycosylation by targeting to different subcellular locations.
► Maximum accumulation was achieved by retention in ER lumen, followed by vacuole and apoplast, cytosolic targeting resulted into degradation.
► Recombinant α1-PI was purified to homogeneity with high yield and biological activity by immunoaffinity chromatography.
► Purified recombinant α1-PI showed compartment-specific N-glycosylation in ER, apoplast and vacuole targeted protein.
► Conformational studies revealed relatively lower stability of the ER-targeted recombinant α1-PI compared to its serum counterpart.