Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, 110016, India.
Microb Cell Fact. 2017 Oct 5;16(1):173. doi: 10.1186/s12934-017-0784-8.
Human serum albumin (HSA)-one of the most demanded therapeutic proteins with immense biotechnological applications-is a large multidomain protein containing 17 disulfide bonds. The current source of HSA is human blood plasma which is a limited and unsafe source. Thus, there exists an indispensable need to promote non-animal derived recombinant HSA (rHSA) production. Escherichia coli is one of the most convenient hosts which had contributed to the production of more than 30% of the FDA approved recombinant pharmaceuticals. It grows rapidly and reaches high cell density using inexpensive and simple subst-rates. E. coli derived recombinant products have more economic potential as fermentation processes are cheaper compared to the other expression hosts. The major bottleneck in exploiting E. coli as a host for a disulfide-rich multidomain protein is the formation of aggregates of overexpressed protein. The majority of the expressed HSA forms inclusion bodies (more than 90% of the total expressed rHSA) in the E. coli cytosol. Recovery of functional rHSA from inclusion bodies is not preferred because it is difficult to obtain a large multidomain disulfide bond rich protein like rHSA in its functional native form. Purification is tedious, time-consuming, laborious and expensive. Because of such limitations, the E. coli host system was neglected for rHSA production for the past few decades despite its numerous advantages.
In the present work, we have exploited the capabilities of E. coli as a host for the enhanced functional production of rHSA (~ 60% of the total expressed rHSA in the soluble fraction). Parameters like intracellular environment, temperature, induction type, duration of induction, cell lysis conditions etc. which play an important role in enhancing the level of production of the desired protein in its native form in vivo have been optimized. We have studied the effect of assistance of different types of exogenously employed chaperone systems on the functional expression of rHSA in the E. coli host system. Different aspects of cell growth parameters during the production of rHSA in presence and absence of molecular chaperones in E. coli have also been studied.
In the present case, we have filled in the gap in the literature by exploiting the E. coli host system, which is fast-growing and scalable at the low cost of fermentation, as a microbial factory for the enhancement of functional production of rHSA, a crucial protein for therapeutic and biotechnological applications.
人血清白蛋白（HSA）是一种需求量最大的治疗性蛋白，具有巨大的生物技术应用价值，它是一种含有 17 个二硫键的大型多功能蛋白。目前 HSA 的来源是人类血浆，这是一种有限且不安全的来源。因此，迫切需要促进非动物来源的重组 HSA（rHSA）生产。大肠杆菌是最方便的宿主之一，它为生产超过 30%的美国食品和药物管理局批准的重组药物做出了贡献。它使用廉价且简单的底物快速生长并达到高细胞密度。与其他表达宿主相比，大肠杆菌衍生的重组产品具有更大的经济潜力，因为发酵过程更便宜。利用大肠杆菌作为富含二硫键的多功能蛋白的宿主的主要瓶颈是表达蛋白的聚集。大多数表达的 HSA 在大肠杆菌细胞质中形成包涵体（超过总表达 rHSA 的 90%）。从包涵体中回收功能性 rHSA 并不受欢迎，因为很难获得功能性天然形式的 rHSA 等大型多功能富含二硫键的蛋白。纯化繁琐、耗时、费力且昂贵。由于这些限制，尽管大肠杆菌具有许多优点，但在过去几十年中，它一直被忽视用于 rHSA 的生产。
在本工作中，我们利用大肠杆菌作为宿主，增强了 rHSA 的功能性生产（约总表达 rHSA 的 60%存在于可溶部分中）。优化了细胞内环境、温度、诱导类型、诱导持续时间、细胞裂解条件等参数，这些参数在体内以天然形式增强所需蛋白的生产水平方面发挥着重要作用。我们研究了不同类型的外源分子伴侣系统对大肠杆菌宿主系统中 rHSA 功能性表达的影响。还研究了在大肠杆菌中存在和不存在分子伴侣时 rHSA 生产过程中细胞生长参数的不同方面。
在本案例中，我们利用大肠杆菌作为宿主系统填补了文献中的空白，大肠杆菌是一种快速生长且可扩展的微生物工厂，其发酵成本低廉，可用于增强治疗性和生物技术应用中关键蛋白 rHSA 的功能性生产。
