Abstract

Tumor necrosis factor-alpha (TNF-α) is an inflammatory cytokine that plays a major role in immune regulation, homeostatic function, and cellular organization. The present study was undertaken to overproduce recombinant human TNF-α (rhTNF-α) in Escherichia coli (E.coli) in high cell density culture. The use of a codon-optimized gene and strong promoter-based (T7) expression system, choice of Terrific Broth (TB) as medium, and subsequent optimization of culture conditions in shake flasks resulted in production of 0.95 g/L insoluble rhTNF-α comprising upto 50% of total cellular protein (TCP) The protein yield further increased upto 1.26 g/L in 1 L TB medium batch culture in bioreactor with the controlled temperature, pH, and dissolved oxygen. In a series of chemostats operated at dilution rates of 0.2 h⁻¹, 0.3 h⁻¹, 0.4 h⁻¹ and 0.5 h⁻¹ the specific growth rate (μ) positively correlated with specific yield (Y_p/x) and a maximum yield of 164 mg/g DCW was obtained at μ = 0.4 h⁻¹ within 4 h post-induction. A fed-batch cultivation in TB with an exponential feeding profile (μ = ∼0.4 h⁻¹) of concentrated feed resulted in an accumulation of 5.5 g/L of rhTNF-α within 14 h of cultivation which accounted for ∼29% of TCP.

抽象的

肿瘤坏死因子-α（TNF-α）是一种炎症性细胞因子，在免疫调节，体内平衡功能和细胞组织中起主要作用。进行本研究是为了在大肠杆菌（E.coli）中过量生产重组人TNF-α（rhTNF-α））在高细胞密度培养中。使用密码子优化的基因和基于强启动子的（T7）表达系统，选择Terrific Broth（TB）作为培养基，随后优化摇瓶中的培养条件，可产生0.95 g / L的不溶性rhTNF-α包含高达总细胞蛋白（TCP）的50％的蛋白质，在温度，pH和溶解氧受控的条件下，在生物反应器中的1 L TB培养基分批培养中，蛋白质产量进一步提高至1.26 g / L。在一系列以0.2ħ稀释速率下操作恒化的^-1，0.3H ^-1，0.4H ^-1和0.5小时^-1的比生长速率（μ）与正相关特定产率（Ý _{p / X}），诱导后4 h内μ = 0.4 h ^-1的最大产量为164 mg / g DCW 。在TB的分批补料培养中，浓缩饲料呈指数补料分布（μ =〜0.4 h ^-1），导致培养14 h内rhTNF-α积累5.5 g / L，约占TCP的29％ 。