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

1605564徐剑锋随着对高价值蛋白质疗法的需求不断增加，植物细胞提供了一个安全且经济高效的替代生物生产平台。然而，蛋白质生产率低是这项技术商业化的常见瓶颈。该项目使植物细胞表达分泌到细胞培养液中的外源蛋白质。这是有利的，因为蛋白质现在被隔离在细胞内的蛋白分解降解中，并且可以很容易地回收和纯化。从这项研究中获得的基础知识将促进我们对植物发育、逆境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者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。具体地说，该项目旨在开发独特的植物Hyp-O-糖基化‘密码’，它精确地指导富含Pro的多肽的O-糖基化，开发新型的Hyp-O-糖基化多肽(HypGPs)，其功能是将连接的异源蛋白排泄到细胞培养基中的分子载体。PI将检验这样一个假设，即经过战略设计的HypGP载体可以通过促进几种模型蛋白质的分泌并稳定培养基中的这些产物来提高植物细胞培养生产率。在烟草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。根据这项研究的结果，将建立在肽序列和链长方面的最优HypGP设计，以实现最高的蛋白质产量。为了评估设计的HypGPs在表达更大和更复杂的治疗蛋白方面的实际和更广泛的应用，将测试两种不同的、具有治疗前景的蛋白质--人α-抗胰蛋白酶(52 KDa)和白介素12(70 KDa)的表达。此外，还将研究Hyp-O-糖基化‘代码’在单子叶水稻细胞培养中工程功能设计者HypGPs的更广泛适用性，以促进我们对单子叶植物中Hyp-O-糖基化的理解，并在水稻细胞培养中实现高蛋白质生产率。该奖项由CBET部门生物技术和生化工程项目共同资助，由分子和细胞生物科学部门系统和合成生物学项目以及刺激竞争研究的实验项目(EPSCoR)共同资助。

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).