Abstract
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.
Highlights
While bacteria are widely used to express recombinant proteins (r-proteins) due to their ease of use, high yield, and low cost, they lack eukaryotic post-translational modification systems[1], which heterologous genes with abundant codons, rarely used in E. coli, may not be efficiently expressed in E. coli and may lead to translation error[2], compromised protein function, improper protein folding, or protein aggregation into inclusion bodies[3,4]
We demonstrated that the novel pHH-GM1 vector consisting of the ctEF-1 first intron in combination with two enhancers (CMV followed by simian virus 40 (SV40)) could be used to express six different genes as well as for co-transfection of cells with multiple plasmids
The alpha kinase 1 (ALPK1) gene was selected for detailed study, and overall, we found that pHH-GM1 vector was superior to six commercial vectors in terms of increased protein expression and green fluorescent protein (GFP) fluorescence, higher yields with two cell types (HEK293-F, CHO-S), higher yields across the expression timeframe, and better cell viability after transfection
Summary
While bacteria are widely used to express recombinant proteins (r-proteins) due to their ease of use, high yield, and low cost, they lack eukaryotic post-translational modification systems[1], which heterologous genes with abundant codons, rarely used in E. coli, may not be efficiently expressed in E. coli and may lead to translation error[2], compromised protein function, improper protein folding, or protein aggregation into inclusion bodies[3,4]. The first intron of EF-1α contains several transcription factors Spl and Apl elements, which seem to have additive effects on its promoter activity[10]. These results showed that both the 5′ flanking region and the first intron of the EF-lα gene are essential for its promoter activity. To identify an efficient expression system that can induce potent expression of various genes, including NLRP3 (inflammasome), ALPK1 (kinase protein), F-actin (motor protein), calmodulin, PP2A (phosphatase), URAT1 (membrane protein), Rab11a (cellular trafficking protein), and myosin IIA (cytoskeleton protein), we systematically inserted various combinations of the C-terminal EF-1α first intron (ctEF-1α) and two translational enhancers (SV40 and CMV) downstream of the SV40 polyA sequence in a CMV promoter-driven gene expression cassette
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