Engineering novel designer glycopeptides as molecular carriers for boosting protein secretion in plant cell culture

小行星1605564 随着对高价值蛋白质治疗药物的需求不断增加，植物细胞提供了一个安全且具有成本效益的替代生物生产平台。然而，低蛋白生产率是该技术商业化的常见瓶颈。该项目设计植物细胞来表达分泌到细胞培养基中的外源蛋白。 这是有利的，因为蛋白质现在与细胞内蛋白水解降解隔离，并且可以容易地回收和纯化。 从这项研究中获得的基础知识将促进我们对植物发育，胁迫防御和植物细胞壁组装的理解。这是一个高度跨学科的研究项目，将为阿肯色州州立大学的研究生和本科生提供独特的学习机会。该项目的总体目标是利用设计师分子载体工程技术开发具有商业竞争力的基于植物细胞的高价值蛋白质生物生产平台。具体而言，该项目旨在利用独特的植物Hyp-O-糖基化“密码”，精确地指导富含脯氨酸的肽的O-糖基化，以开发新型Hyp-O-糖基化肽（HypGPs），其作为分子载体在分泌连接的异源蛋白到细胞培养基中发挥作用。PI将测试以下假设：战略设计的HypGP载体可以通过促进几种模型蛋白的分泌并在培养基中稳定这些产品来提高植物细胞培养生产力。烟草BY-2细胞中精确的Hyp-O-糖基化过程，以及促进蛋白质分泌的关键机制将被剖析。 基于本研究的结果，将建立在肽序列和链长度方面的最佳HypGP设计，以实现最高的蛋白质产量。为了评估设计师HypGP用于表达更大和更复杂的治疗性蛋白质的实际和更广泛的应用，将测试两种不同的和有治疗前景的蛋白质，人α 1-抗胰蛋白酶（52 kDa）和白细胞介素-12（70 kDa）的表达。此外，本发明还本研究旨在探索Hyp-O-糖基化"密码"在单子叶植物水稻细胞培养中工程化功能设计HypGPs的更广泛应用，以促进我们对单子叶植物Hyp-O-糖基化的理解，并在水稻细胞培养中实现高蛋白生产率。由分子和细胞生物科学部的系统和合成生物学计划以及刺激竞争研究的实验计划（EPSCoR）资助。

1605564 Xu, Jianfeng With increasing needs for high-value protein-based therapeutics, plant cells offer a safe and cost-effective alternative bioproduction platform. However, low protein productivity is a common bottleneck in the commercialization of this technology. This project engineers plant cells to express foreign proteins that are secreted into the cell culture medium. This is advantageous as the protein is now sequestered from intracellular proteolytic degradation and can be readily recovered and purified. Fundamental knowledge gained from this research will advance our understanding of plant development, stress defense, and plant cell wall assembly. This is a highly interdisciplinary research project that will present unique learning opportunities for both graduate and undergraduate students at Arkansas State University.The overall goal of this project is to leverage a designer molecular carrier engineering technology in developing a commercially competitive plant cell-based bioproduction platform for high-value proteins. Specifically, this project aims to exploit the unique plant Hyp-O-glycosylation 'code' that precisely directs the O-glycosylation of a proline-rich peptide, to develop novel Hyp-O-glycosylated peptides (HypGPs) that function as a molecular carrier in excreting conjoined heterologous proteins to cell culture media. The PIs will test the hypothesis that strategically designed HypGP carriers can function to improve plant cell culture productivity by boosting the secretion of several model proteins and stabilizing these products in culture media. The precise Hyp-O-glycosylation process in tobacco BY-2 cells, and the key mechanisms facilitating protein secretion will be dissected. Based on the outcome of this study, optimal HypGP design in terms of peptide sequence and chain length to achieve the highest protein yields will be established. In order to assess the practical and broader applications of the designer HypGPs for expressing larger and complex therapeutic proteins, expression of two distinct and therapeutically promising proteins, human alfa1-antitrypsin (52 kDa) and interleukin-12 (70 kDa) will be tested. Additionally, broader applicability of Hyp-O-glycosylation 'code' to engineering functional designer HypGPs in monocot rice cell culture will be investigated towards advancing our understanding about the Hyp-O-glycosylation in monocot plants and achieving high protein productivity in rice cell culture.This award by the Biotechnology and Biochemical Engineering Program of the CBET Division is co-funded by the Systems and Synthetic Biology Program of the Division of Molecular and Cellular Biosciences and by the Experimental Program to Stimulate Competitive Research (EPSCoR).