High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells
High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells
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DOI：
10.1093/nar/30.2.e9
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发表时间：
2002-01-15
影响因子：
14.9
通讯作者：
Kamen, A
中科院分区：
文献类型：
--
作者：
Durocher, Y;Perret, S;Kamen, A
本文报道了一种用聚乙烯亚胺（PEI）转染悬浮培养的人胚肾293细胞系的方法。使用绿色荧光蛋白（GFP）和人胎盘分泌的碱性磷酸酶（SEAP）作为报告基因来监测转染效率和生产力。使用线性或分支的25 kDa PEI获得高达75%的GFP阳性细胞。检测了293细胞系和表达SV 40大T抗原（293 T）或EB病毒（EBV）EBNA 1蛋白（293 E）的两种遗传变体的蛋白表达。使用含有EBV oriP的质粒pCEP 4，用293 E细胞获得最高表达水平。我们设计了pTT载体，这是一种基于oriP的载体，具有改进的巨细胞病毒表达盒。与pcDNA3.1和pCEP 4载体相比，使用该载体，在293 E细胞中SEAP表达分别增加了10倍和3倍。血清的存在对基因转移和表达有积极影响。用线性PEI转染悬浮生长细胞更有效，并且不受条件培养基存在24 h的影响。使用pTT载体，从3.5升生物反应器回收>20 mg/l纯化的His标记的SEAP。细胞内蛋白质也以高达50 mg/l的水平产生，占总细胞蛋白质的20%。
A scalable transfection procedure using polyethylenimine (PEI) Is described for the human embryonic kidney 293 cell line grown in suspension. Green fluorescent protein (GFP) and human placental secreted alkaline phosphatase (SEAP) were used as reporter genes to monitor transfection efficiency and productivity. Up to 75% of GFP-positive cells were obtained using linear or branched 25 kDa PEI. The 293 cell line and two genetic variants, either expressing the SV40 large T-antigen (293T) or the Epstein-Barr virus (EBV) EBNA1 protein (293E), were tested for protein expression. The highest expression level was obtained with 293E cells using the EBV oriP-containing plasmid pCEP4. We designed the pTT vector, an oriP-based vector having an improved cytomegalovIrus expression cassette. Using this vector, 10- and 3-fold increases in SEAP expression was obtained in 293E cells compared with pcDNA3.1 and pCEP4 vectors, respectively. The presence of serum had a positive effect on gene transfer and expression. Transfection of suspension-g rowing cells was more efficient with linear PEI and was not affected by the presence of medium conditioned for 24 h. Using the pTT vector, >20 mg/l of purified His-tagged SEAP was recovered from a 3.5 l bioreactor. Intracellular proteins were also produced at levels as high as 50 mg/l, representing up to 20% of total cell proteins.