Plant Transgenic Lab, CSIR-National Botanical Research Institute, Rana Pratap Marg, P.O. Box 436, Lucknow 226001, India.
Plant Sci. 2012 Nov;196:53-66. doi: 10.1016/j.plantsci.2012.07.004. Epub 2012 Jul 28.
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.
通过将治疗性重组人蛋白水解酶抑制剂（α₁-PI）靶向到不同的亚细胞位置，如内质网（ER）、质外体、液泡和细胞质，分析了蛋白质积累部位对产量、生物活性和体内稳定性的影响，这些位置在转基因番茄植物的叶片中。通过免疫组织化学染色监测重组 α₁-PI 蛋白在转基因植物细胞中的原位定位。通过保留在 ER 腔中，在 T₀ 和 T₁ 代转基因番茄植物中，重组 α₁-PI 最大积累量达到总可溶性蛋白（TSP）的 1.5%至 3.2%，其次是液泡和质外体，而细胞质靶向则导致蛋白质降解。通过残留猪胰弹性蛋白酶（PPE）活性测定和带位移测定监测，植物来源的重组 α₁-PI 显示出弹性酶抑制的生物活性。从转基因番茄植物中以高产、均一性和生物活性纯化重组 α₁-PI。纯化的蛋白质在 SDS-PAGE 上呈现出约 48-50 kDa 的单带，等电点（pI）值在 5.1 和 5.3 之间。纯化的重组 α₁-PI 的质谱和光谱学研究结果表明，与天然血清 α₁-PI 相比，重组蛋白的结构完整性相当。用纯化的重组 α₁-PI 进行酶解糖基化和凝集素结合试验表明，靶向内质网、质外体和液泡的蛋白质具有特定的细胞区室 N-糖基化。基于尿素诱导变性和圆二色性（CD）光谱的构象研究表明，与天然血清 α₁-PI 相比，重组 α₁-PI 蛋白的稳定性相对较低。通过静脉途径在大鼠中对植物来源的重组和人血浆纯化的 α₁-PI 进行药代动力学评估，表明重组蛋白的血浆清除速度明显更快，曲线下面积（AUC）更低。我们的数据表明了蛋白质分选序列的重要性，并证实了使用转基因植物生产稳定、糖基化和具有生物活性的重组 α₁-PI 用于进一步治疗应用的可行性。
