抗调亡基因bcl-xl过表达提高工程细胞IVCC及目的蛋白表达的研究

用无血清培养基或化学成分明确的培养基生产治疗用重组蛋白已成为趋势。然而,在此条件下凝血因子、糖蛋白激素等微量糖蛋白的表达十分困难,其主要原因之一是在细胞培养过程中工程细胞大量凋亡造成的细胞密度低和生存期短。通过将早期抗凋亡基因导入工程细胞并进行过表达可改善工程细胞的活细胞密度积分（integral viable cell concentration,IVCC）,提高表达量。该研究将bcl-xl基因导入工程细胞,筛选其高表达细胞株,并验证工程细胞的抗凋亡能力,获得了稳定表达抗凋亡蛋白和目的蛋白的工程细胞株。与母细胞相比,稳定表达Bcl－xL的工程细胞的IVCC提高了50％,最终目的蛋白表达增加超过200％,显示抗凋亡基因bcl-xl的过表达可改善工程细胞在无血清悬浮培养过程中的细胞凋亡,提高表达量,为表达人凝血因子、糖蛋白激素等微量糖蛋白奠定了基础。     

用于生产重组蛋白药物的抗凋亡CHO宿主细胞株的建立

哺乳动物工程细胞在大规模培养生产重组蛋白时很容易发生细胞凋亡,从而导致生产过程提前终止,造成生产成本高昂。细胞代谢产物氨已被证明可以促进细胞凋亡,而线粒体膜整合蛋白Bcl-2可以通过促进线粒体膜完整性而抑制细胞凋亡。本实验应用谷氨酰胺合成酶加压系统在CHO工程细胞中高效表达中国仓鼠Bcl-2蛋白,使细胞具有抗凋亡能力的同时,利用谷氨酸和氨合成谷氨酰胺而有效降低培养基中氨的含量,从而达到抑制细胞凋亡的目的。     

哺乳动物细胞培养生产药用蛋白的关键环节

近年来,应用哺乳动物工程细胞系统生产药用蛋白显示了越业越重要的地位,本重点介绍了当前如何在生产药用蛋白的哺乳动物工程细胞表达系统,培养、生产技术,认证,生产过程组织等关键环节上进行优化,从而达到高效,安全、经济生产模式的核心技术和策略。     

腺病毒E1B-19K基因过表达对HEK293细胞生长和代谢的影响

细胞凋亡是动物细胞大规模培养中影响活细胞密度和目的产品质量的重要因素,过表达抗凋亡基因是目前常用的提高工程细胞凋亡抗性的一种策略。拟在HEK293细胞中过表达腺病毒E1B-19K基因,挑取了不同E1B-19K表达水平的单克隆细胞,考察在不同培养条件下细胞的凋亡水平和代谢情况。E1B-19K的过表达可显著增强细胞在低葡萄糖、低血清和无谷氨酰胺3种培养条件下的抗凋亡能力,使凋亡细胞比例降低60%~80%;E1B-19K的过表达可使批次培养HEK293细胞的衰退期延迟2天,而对细胞的葡萄糖、乳酸和谷氨酰胺等的代谢无显著影响。结果表明,过表达E1B-19K是一种有效减缓HEK293细胞在培养过程中凋亡的策略。     

基因编辑在构建抗凋亡工程细胞中的应用

21世纪以来,人们在生物科技领域取得了长足进步。一方面,基因编辑技术的问世为精确修饰目的基因带来可能,即能够在活细胞中完成特定DNA片段的插入、删除、替换、激活、抑制等任务;另一方面,越来越多的新型重组蛋白药物被开发并应用于对抗肿瘤等人类重大疾病,相比于传统化学药物,它具有高特异性和低副作用等显著优势,治疗效果得到普遍认可。目前用于生产治疗性重组蛋白的工程细胞株主要来源于哺乳动物,然而,由于生产过程中任何环境因素的改变都可能使工程细胞凋亡,严重影响蛋白的表达水平,因此大大提高了生产成本。研究人员在明确工程细胞的生长与死亡相关机制后,利用基因编辑技术对其进行了定向改造,提高了其表达水平。该文就对该方面研究成果进行了综述。     

利用哺乳动物细胞表达外源蛋白的研究进展

利用哺乳动物细胞表达外源蛋白已广泛应用于生物产品的制备,哺乳动物细胞是表达具有天然活性蛋白的最佳宿主,且具有易被转染,遗传稳定,产物可分泌表达,并易于纯化和大规模生产等方面的优势。本文对选择载体类型,载体元件（包括启动子,增强子,选择标记等）以及在哺乳动物细胞大规模培养过程中培养环境,细胞凋亡的抑制和控制细胞增殖和基因表达的Tet-switch系统等方面的进展作一综述,以探讨提高该系统表达产量的有效方法,更好地应用于生物制品的生产。     

用于重组蛋白生产的哺乳动物细胞表达新技术

近年来,用于重组蛋白生产的哺乳动物细胞表达领域涌现出一系列革命性的新技术。优化的工程细胞为表达重组蛋白提供了优良的宿主;基于荧光的筛选方法可以快捷地得到高表达细胞株;高通量的培养工艺能够预测适合外源蛋白表达的细胞培养条件;可抛弃式生物反应器为大规模细胞培养提供了更多的选择;大规模瞬时表达技术节省了重组蛋白的生产时间。这些新技术提高了重组蛋白的研发和生产效率,加快了蛋白药物的工业化进程。     

通过高表达Igf1/Bcl-2或Bcl-2/Cyclin E基因组合使CHO细胞适于在无蛋白培养基中抗凋亡培

哺乳动物细胞表达系统是生产重组蛋白药物最常用的表达系统。但在无蛋白培养基中,哺乳动物细胞生长活力差,且容易发生细胞凋亡,因而难以大规模培养。为解决此问题,应用双顺反子表达载体在CHO-dhfr-细胞中同时表达Igf-1/Bcl-2或Bcl-2/Cyclin E基因组合,通过Bcl-2使细胞获得抗凋亡能力;通过Igf-1或Cyclin E促进细胞生长分裂,使细胞获得在无蛋白培养基中生长的能力。以上述基因组合转染CHO-dhfr^-细胞,应用Western blot从G418抗性克隆中分别筛选到Bcl-2高表达克隆若干个,对其中表达Bcl-2最高的CHO-IB3和CHO-BC1做进一步Western blot和流式细胞分析,确认此两个细胞株分别高表达Igf-1/Bcl-2和Bcl^-2/Cyclin E基因组合。分别通过撤去血清和加入放线菌素D诱导细胞凋亡,并以流式细胞术和DNA Ladder法检测细胞凋亡,证明CHO-IB3和CHO-BC1均具有较强的抗细胞凋亡能力。MTT法证明两个细胞株在不含血清的IMDM培养基中的增殖活力显著高于CHO-dhfr-对照细胞。在细胞培养瓶中的连续培养实验表明,CHO-IB3和CHO-BC1在本实验室设计的IMEM无蛋白培养基中的生长速度和活细胞数显著高于CHOdhfr-对照细胞。提示此两个细胞系能够在无血清培养基中抗凋亡高活力生长,适于作为生物工程宿主细胞。     

通过高表达Igf1/Bcl-2或Bcl-2/Cyclin E基因组合使CHO细胞适于在无蛋白培养基中抗凋亡培养

哺乳动物细胞表达系统是生产重组蛋白药物最常用的表达系统。但在无蛋白培养基中,哺乳动物细胞生长活力差,且容易发生细胞凋亡,因而难以大规模培养。为解决此问题,应用双顺反子表达载体在CHO-dhfr^-细胞中同时表达Igf-1／Bcl-2或Bcl-2／CyclinE基因组合,通过Bcl-2使细胞获得抗凋亡能力;通过1gf-1或CyclinE促进细胞生长分裂,使细胞获得在无蛋白培养基中生长的能力。以上述基因组合转染CHO-dhfr^-细胞,应用Western blot从G418抗性克隆中分别筛选到Bcl-2高表达克隆若干个,对其中表达Bcl-2最高的CHO-IB3和CHO-Bcl做进一步Western blot和流式细胞分析,确认此两个细胞株分别高表达Igf-1／Bcl-2和Bcl-2／CyclinE基因组合。分别通过撤去血清和加入放线菌素D诱导细胞凋亡,并以流式细胞术和DNA Ladder法检测细胞凋亡,证明CHO-IB3和CHO一BCl均具有较强的抗细胞凋亡能力。MTT法证明两个细胞株在不含血清的IMDM培养基中的增殖活力显著高于CHO-dhfr^-对照细胞。在细胞培养瓶中的连续培养实验表明,CHO-IB3和CHO-BCl在本实验室设计的IMEM无蛋白培养基中的生长速度和活细胞数显著高于CHO-dhfr^-对照细胞。提示此两个细胞系能够在无血清培养基中抗凋亡高活力生长,适于作为生物工程宿主细胞。     

热休克蛋白27 在肿瘤中的研究进展

热休克蛋白27(HSP27)作为热应激蛋白表达于各种生理或是环境损伤之后,保护细胞生存,其具有多种功能包括:分子伴侣,抗凋亡,参与细胞运动等。近年来发现HSP27在多种肿瘤中过度表达,参与肿瘤的发生发展,分化,耐药以及转移等方面,因而抑制HSP27成为一种新的肿瘤治疗策略。本文就相关研究进展进行综述。     

Links between metabolism and apoptosis in mammalian cells: applications for anti-apoptosis engineering

Production of complex recombinant proteins requires the culture of mammalian cells in bioreactors. Inherent in these cultures is the problem of cell death, which can result from nutrient depletion, byproduct accumulation, and other bioreactor stresses which signal the cell to die through apoptosis, or programmed cell death. Apoptosis is a highly regulated pathway of both pro- and anti-apoptotic proteins that promote cell survival or death, and cell engineering efforts to inhibit the apoptosis pathway have led to increased culture viability and recombinant protein production. Originally, the exclusive function of many of these pathway proteins was believed to be binding at the mitochondria and regulating apoptosis through modulation of the mitochondria permeability. While this protein functionality does still hold true, it is now evident that these proteins also include roles in the metabolic processes of the mitochondria. Furthermore, apoptosis pathway proteins in other organelles within the cell may also both modulate apoptosis and metabolism. This review first details the known links that exist between apoptosis proteins and metabolic functions in the cytosol, mitochondria, and endoplasmic reticulum. Second, the review turns to look at potentially new cell engineering strategies that are linked to metabolism for improving cell culture viability and protein production.     

Bioreactor engineering for recombinant protein production in plant cell suspension cultures

A review of over 15 years of research, development and commercialization of plant cell suspension culture as a bioproduction platform is presented. Plant cell suspension culture production of recombinant products offers a number of advantages over traditional microbial and/or mammalian host systems such as their intrinsic safety, cost-effective bioprocessing, and the capacity for protein post-translational modifications. Recently significant progress has been made in understanding the bottlenecks in recombinant protein expression using plant cells, including advances in plant genetic engineering for efficient transgene expression and minimizing proteolytic degradation or loss of functionality of the product in cell culture medium. In this review article, the aspects of bioreactor design engineering to enable plant cell growth and production of valuable recombinant proteins is discussed, including unique characteristics and requirements of suspended plant cells, properties of recombinant proteins in a heterologous plant expression environment, bioreactor types, design criteria, and optimization strategies that have been successfully used, and examples of industrial applications.     

Anti-apoptosis engineering

An increased understanding of apoptosis makes anti-apoptosis engineering possible, which is an approach used to inhibit apoptosis for the purpose of therapeutic, or industrial applications in the treatment of the diseases associated with increased apoptosis, or to improve the productivity of animal cell cultures, respectively. Some known anti-apoptotic proteins are the Bcl-2 family, IAP (inhibitor of apoptosis) and Hsps (heat shock proteins), with which anti-apoptosis engineering has progressed. This article reviews anti-apoptosis engineering using known anti-apoptotic compounds, and introduces a 30 K protein, isolated from silkworm hemolymph, as a novel anti-apoptotic protein, that shows no homology with other known anti-apoptotic proteins. The regulation of apoptosis, using anti-apoptotic proteins and genes originating from the silkworm,Bombyx mori, may provide a new strategy in this field.     

Expression and subcellular targeting of human insulin-like growth factor binding protein-3 in transgenic tobacco plants

Human insulin-like growth factor binding protein-3 (hIGFBP-3) is a multifunctional protein which has high affinity for insulin-like growth factor-I (IGF-I). It combines with IGF-I to form a tertiary complex in circulation, thus regulating the activity of IGF-I. Furthermore, recombinant hIGFBP-3 (rhIGFBP-3) has been found to negatively regulate cell proliferation and induce apoptosis. In this study, we have established an efficient plant bioreactor platform for mass production of rhIGFBP-3. Different expression constructs, driven by the seed-specific phaseolin promoter, were designed and transformed into tobacco plant via Agrobacterium. To enhance protein expression level, the signal peptide (SP) and the C-terminal tetrapeptide AFVY of phaseolin were used to direct rhIGFBP-3 to protein storage vacuole (PSV) in tobacco seed for stable accumulation. Western blot analysis showed that rhIGFBP-3 was successfully synthesized in transgenic tobacco seeds, with the highest protein expression of 800 μg/g dry weight. The localization of rhIGFBP-3 in PSV was also evident by confocal immunofluorescence microscopy. Our results indicated that protein sorting sequences could benefit the expression level of rhIGFBP-3 and it is feasible to use plant as “bio-factory” to produce therapeutic recombinant proteins in large quantity.     

Enhancement of mussel adhesive protein production in Escherichia coli by co-expression of bacterial hemoglobin

Mussel adhesive proteins (MAPs) have been considered as potential underwater and medical bioadhesives. Previously, we reported a functional expression of recombinant MAP hybrid fp-151, which is a fusion protein with six type 1 (fp-1) decapeptide repeats at each type 5 (fp-5) terminus, with practical properties in Escherichia coli. In the present work, we introduced the Vitreoscilla hemoglobin (VHb) co-expression strategy to enhance the production levels of hybrid fp-151 since VHb has been successfully used for efficient oxygen utilization in several expression systems, including E. coli. In both batch-type flask and fed-batch-type bioreactor cultures, we found that co-expression of VHb conferred higher cell growth and hybrid fp-151 production. Its positive effects were significantly increased in high cell density bioreactor cultures as the microaerobic environment was more quickly and severely formed. We obtained a approximately 1.9-fold higher (approximately 1 g/L) production of MAP fp-151 from VHb co-expressing cells in fed-batch bioreactor cultures as compared to that from VHb non-expressing cells. Collectively and regardless of the culture type, VHb co-expression strategy was successful in enhancing the production of recombinant mussel adhesive proteins in the E. coli expression system.     

Kinetics and modeling of GM-CSF production by recombinant yeast in a three-phase fluidized bed bioreactor

Continuous production of a recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) by Saccharomyces cerevisiae strain XV2181 (a/a, Trp 1) containing plasmid palphaADH2 and immobilized on porous glass beads in a fluidized bed bioreactor was studied. Kinetic models for plasmid stability, cell growth, and protein production in the three-phase fluidized bed bioreactor were developed and used to study the effects of solid loading or cell immobilization on plasmid stability and recombinant protein production. With increasing cell immobilization or solid loading in the bioreactor, plasmid stability and protein production improved significantly. The improvements could be attributed to the decreased theta value, which is the plasmid loss probability during cell division and is an indication of segregational instability of the recombinant cell, and the increased alpha value, which is the ratio of the specific growth rate of a plasmid-carrying cell to that of a plasmid-free cell and is indicative of competitive stability of the recombinant cell culture. theta decreased from 0.552 to 0.042 and alpha increased from 0.351 to 0.991 when solid loading in the bioreactor was increased from 5% (v/v) to 33%. The model simulation also showed that the specific growth rate of cells in the bioreactor was lower at higher solid loading. This indicated that there was significant mass transfer limitation, particularly for oxygen transfer, when the total cell density in the bioreactor was high at high solid loading. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 470-477, 1997.     

Regulation of Bcl-2-family proteins in myeloma cells by three myeloma survival factors: interleukin-6, interferon-alpha and insulin-like growth factor 1

As survival regulation is a key process in multiple myeloma biology, we have studied the Bcl-2 family proteins that can be regulated by three myeloma cell survival factors: interleukin-6 (IL-6), interferon-alpha (IFN-alpha) and insulin-like growth factor (IGF-1). Eleven myeloma cell lines, whose survival and proliferation are dependent on addition of IL-6, variably expressed 10 anti-apoptotic or pro-apoptotic proteins of the Bcl-2-family. When myeloma cells from four cell lines were IL-6 starved and activated with IL-6 or IFN-alpha, we observed that only Mcl-1 expression was up-regulated with myeloma cell survival induction. Nor was obvious regulation of these 10 pro-apoptotic or anti-apoptotic proteins found with IGF-1, another potent myeloma cell survival factor. Our results indicate that the myeloma cell survival activity of IL-6 linked to Bcl-xL regulation cannot be generalized and emphasize that Mcl-1 is the main target of IL-6 and IFN-alpha stimulation. However, other changes in the activity of the Bcl-2 protein family or other apoptosis regulators must be identified to elucidate the IGF-1 action mechanism. Cell Death and Differentiation (2000) 7, 1244 - 1252.     

‘Omics driven discoveries of gene targets for apoptosis attenuation in CHO cells

Chinese hamster ovary (CHO) cells are widely used in biopharmaceutical production. Improvements to cell lines and bioprocesses are constantly being explored. One of the major limitations of CHO cell culture is that the cells undergo apoptosis, leading to rapid cell death, which impedes reaching high recombinant protein titres. While several genetic engineering strategies have been successfully employed to reduce apoptosis, there is still room to further enhance CHO cell lines performance. ‘Omics analysis is a powerful tool to better understand different phenotypes and for the identification of gene targets for engineering. Here, we present a comprehensive review of previous CHO 'omics studies that revealed changes in the expression of apoptosis‐related genes. We highlight targets for genetic engineering that have reduced, or have the potential to reduce, apoptosis or to increase cell proliferation in CHO cells, with the final aim of increasing productivity.