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

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...

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Autores principales: Lee, Chi-Pin, Ko, Albert Min-Shan, Chiang, Shang-Lun, Lu, Chi-Yu, Tsai, Eing-Mei, Ko, Ying-Chin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6193983/
https://www.ncbi.nlm.nih.gov/pubmed/30337625
http://dx.doi.org/10.1038/s41598-018-33500-0
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author Lee, Chi-Pin
Ko, Albert Min-Shan
Chiang, Shang-Lun
Lu, Chi-Yu
Tsai, Eing-Mei
Ko, Ying-Chin
author_facet Lee, Chi-Pin
Ko, Albert Min-Shan
Chiang, Shang-Lun
Lu, Chi-Yu
Tsai, Eing-Mei
Ko, Ying-Chin
author_sort Lee, Chi-Pin
collection PubMed
description 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.
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spelling pubmed-61939832018-10-24 Regulatory elements in vectors containing the ctEF-1α first intron and double enhancers for an efficient recombinant protein expression system Lee, Chi-Pin Ko, Albert Min-Shan Chiang, Shang-Lun Lu, Chi-Yu Tsai, Eing-Mei Ko, Ying-Chin Sci Rep Article 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. Nature Publishing Group UK 2018-10-18 /pmc/articles/PMC6193983/ /pubmed/30337625 http://dx.doi.org/10.1038/s41598-018-33500-0 Text en © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Lee, Chi-Pin
Ko, Albert Min-Shan
Chiang, Shang-Lun
Lu, Chi-Yu
Tsai, Eing-Mei
Ko, Ying-Chin
Regulatory elements in vectors containing the ctEF-1α first intron and double enhancers for an efficient recombinant protein expression system
title Regulatory elements in vectors containing the ctEF-1α first intron and double enhancers for an efficient recombinant protein expression system
title_full Regulatory elements in vectors containing the ctEF-1α first intron and double enhancers for an efficient recombinant protein expression system
title_fullStr Regulatory elements in vectors containing the ctEF-1α first intron and double enhancers for an efficient recombinant protein expression system
title_full_unstemmed Regulatory elements in vectors containing the ctEF-1α first intron and double enhancers for an efficient recombinant protein expression system
title_short Regulatory elements in vectors containing the ctEF-1α first intron and double enhancers for an efficient recombinant protein expression system
title_sort regulatory elements in vectors containing the ctef-1α first intron and double enhancers for an efficient recombinant protein expression system
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6193983/
https://www.ncbi.nlm.nih.gov/pubmed/30337625
http://dx.doi.org/10.1038/s41598-018-33500-0
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