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

设计新颖的糖肽作为分子载体，促进植物细胞培养中的蛋白质分泌

• 批准号: 1605564
• 负责人: Jianfeng Xu
• 金额: $ 44.32万
• 依托单位: Arkansas State University Main Campus
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1605564&HistoricalAwards=false
• 关键词: Engineering; novel; designer; glycopeptides; molecular

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).

随着人们对高价值蛋白质治疗药物的需求不断增加，植物细胞提供了一种安全、经济的替代生物生产平台。然而，蛋白质产量低是该技术商业化的一个常见瓶颈。这个项目的工程师种植细胞来表达分泌到细胞培养基中的外来蛋白质。这是有利的，因为蛋白质现在从细胞内蛋白水解降解中分离出来，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。具体来说，该项目旨在利用独特的植物hypo -糖基化“代码”，精确地指导富含脯氨酸的肽的o -糖基化，开发新型的hypp - o -糖基化肽（hypgp），作为分子载体将连接的异源蛋白排泄到细胞培养基中。这些pi将验证这样一个假设，即战略性设计的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物来提高植物细胞培养效率。烟草BY-2细胞中hypo -糖基化的精确过程，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，将在肽序列和链长方面建立最优的HypGP设计，以实现最高的蛋白产量。为了评估设计HypGPs在表达更大、更复杂的治疗性蛋白方面的实际和更广泛的应用，将测试两种不同的、有治疗前景的蛋白的表达，人类α -1 -抗胰蛋白酶（52 kDa）和白细胞介素-12 （70 kDa）。此外，hypo -糖基化“代码”在单株水稻细胞培养中更广泛的适用性将被研究，以促进我们对单株植物hypo -糖基化的理解，并在水稻细胞培养中实现高蛋白质产量。该奖项由CBET部门的生物技术和生化工程项目颁发，由分子和细胞生物科学部的系统和合成生物学项目以及刺激竞争性研究的实验项目（EPSCoR）共同资助。

项目成果

Dramatic secretion of recombinant protein expressed in tobacco cells with a designer glycopeptide tag is highly impacted by medium composition

带有设计糖肽标签的烟草细胞中表达的重组蛋白的显着分泌受到培养基成分的高度影响

• DOI: 10.1007/s00299-016-2051-6
• 发表时间: 2016
• 期刊: Plant Cell Reports
• 影响因子: 6.2
• 作者: Zhang, Ningning;Dolan, Maureen;Wu, Di;Phillips, Gregory C.;Xu, Jianfeng
• 通讯作者: Xu, Jianfeng