title: Differential subcellular targeting of recombinant human α₁-proteinase inhibitor influences yield, biological activity and in planta stability of the protein in transgenic tomato plants.
abstract: The response of protein accumulation site on yield, biological activity and in planta stability of therapeutic recombinant human 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.
authors.affiliation: Plant Transgenic Lab, CSIR-National Botanical Research Institute, Rana Pratap Marg, P.O. Box 436, Lucknow 226001, India.
authors.forename: Shweta
authors.initials: S
authors.lastname: Jha
authors.forename: Saurabh
authors.initials: S
authors.lastname: Agarwal
authors.forename: Indraneel
authors.initials: I
authors.lastname: Sanyal
authors.forename: G K
authors.initials: GK
authors.lastname: Jain
authors.forename: D V
authors.initials: DV
authors.lastname: Amla
mesh_terms: D000818:Animals; D019540:Area Under Curve; D001483:Base Sequence; D015153:Blotting, Western; D015180:Electrophoresis, Gel, Two-Dimensional; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005240:Feasibility Studies; D006031:Glycosylation; D006801:Humans; D007150:Immunohistochemistry; D042541:Intracellular Space / Q000378:metabolism; D007700:Kinetics; D018551:Solanum lycopersicum / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D055550:Protein Stability; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism* / Q000493:pharmacokinetics / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013552:Swine; D014617:Vacuoles / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin; D010196:Pancreatic Elastase