Regulatory elements in vectors containing the ctEF-1α first intron and double enhancers for an efficient recombinant protein expression system

• Published in: Scientific reports Vol. 8; no. 1; pp. 15396 - 9
• Main Authors: Lee, Chi-Pin, Ko, Albert Min-Shan, Chiang, Shang-Lun, Lu, Chi-Yu, Tsai, Eing-Mei, Ko, Ying-Chin
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 18.10.2018; Nature Publishing Group
• Subjects: 42/44; 631/208/199; 631/61/185; 82; 82/80; 82/83; Actin; Animals; CHO Cells; Cricetinae; Cricetulus; Cytomegalovirus; Cytomegalovirus - genetics; Enhancer Elements, Genetic - genetics; Enhancers; Gene Expression; Genetic Vectors - genetics; Green Fluorescent Proteins - chemistry; Green Fluorescent Proteins - genetics; HEK293 Cells; Humanities and Social Sciences; Humans; Introns - genetics; Kinases; multidisciplinary; Myosin; Peptide Elongation Factor 1 - chemistry; Peptide Elongation Factor 1 - genetics; Plasmids; Plasmids - chemistry; Protein Domains - genetics; Proteins; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Regulatory sequences; Regulatory Sequences, Nucleic Acid - genetics; Science; Science (multidisciplinary); Supplements; Transfection; Transfection - methods; Kinase Alpha (ALPK1); Double Enhancer; HEK293T Cells; Simian Virus (SV40); Commercial Plasmid
• DOI: 10.1038/s41598-018-33500-0
• ISSN: 2045-2322

To establish a stable and scalable transient protein production system, we modified the EF-1 first intron size and verified the order of two recombinant enhancers downstream of the SV40 polyA sequence. This new vector was named pHH-Gemini (pHH-GM1) and was used to express alpha kinase 1 ( ALPK1 ) and various other proteins, NLRP3, F-actin, Camodulin, PP2A, URAT1, Rab11a and myosin IIA. The results showed that, compared with six commercial plasmids, pHH-GM1 significantly enhanced His-HA-ALPK1 expression in a western blot analysis of transfected HEK293T cells. The expression of various other genes was also successful using the pHH-GM1 vector. In addition, we inserted turbo green florescence protein (tGFP) into the pHH-GM1 vector, and an improvement in fluorescence intensity was observed after transient transfection of HEK293T cells. For large-scale production, protein production was tested by standard supplementation with one volume of medium, and volumetric yields of 2 and 2.3 mg/L were achieved with pHH-GM1-ALPK1 in HEK293-F and CHO-S cells, respectively. We found that cell viability was more than 70% 11 days after cells were transfected with the pHH-GM1 vector. The pHH-GM1 vector with the ctEF-1α first intron and double enhancers, Simian virus 40 and Cytomegalovirus (SV40 and CMV) is an efficient CMV promoter-based gene expression system that can potentially be applied to study genes of interest and improve protein production.