초록
The response of protein accumulation site on yield, biological activity and in planta stability of therapeutic recombinant human α<SUB>1</SUB>-proteinase inhibitor (α<SUB>1</SUB>-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 α<SUB>1</SUB>-PI protein in transgenic plant cells was monitored by immunohistochemical staining. Maximum accumulation of recombinant α<SUB>1</SUB>-PI in T<SUB>0</SUB> and T<SUB>1</SUB> 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 α<SUB>1</SUB>-PI showed biological activity for elastase inhibition, as monitored by residual porcine pancreatic elastase (PPE) activity assay and band-shift assay. Recombinant α<SUB>1</SUB>-PI was purified from transgenic tomato plants with high yield, homogeneity and biological activity. Purified protein appeared as a single band of ~48-50kDa on SDS-PAGE with pI value ranging between 5.1 and 5.3. Results of mass spectrometry and optical spectroscopy of purified recombinant α<SUB>1</SUB>-PI revealed the structural integrity of the recombinant protein comparable to native serum α<SUB>1</SUB>-PI. Enzymatic deglycosylation and lectin-binding assays with the purified recombinant α<SUB>1</SUB>-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 α<SUB>1</SUB>-PI protein, compared to its serum counterpart. Pharmacokinetic evaluation of plant derived recombinant and human plasma-purified α<SUB>1</SUB>-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 α<SUB>1</SUB>-PI for further therapeutic applications.