Genomic Analysis and Isolation of RNA Polymerase II Dependent Promoters from Spodoptera frugiperda

Bleckmann, Maren; Fritz, Markus Hsi Yang; Bhuju, Sabin; Jarek, Michael; Schürig, Margitta; Geffers, Robert; Beneš, Vladimı́r; Besir, Hüseyin; Heuvel, Joop van den

doi:10.1371/journal.pone.0132898

Cited by 35 publications

(33 citation statements)

References 41 publications

(46 reference statements)

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“…This observation meets the expectation as it is probably caused by the overall lower plasmid based protein production in Sf21 cells (Bleckmann et al, 2015). In conclusion the results indicate that the SplitGFP screen is functional in Hi5 cells and expression can be monitored with the automated BioLector Multicultivation System.…”

Section: Resultssupporting

confidence: 89%

“…The DNA sequences of spl GFP1-10 and spl GFP11 were designed according to Waldo et al (1999) (Cabantous et al, 2005b) and synthesized by Genscript (Piscataway, NJ). The synthesized constructs as well as the vector OpIE2-eGFP (Bleckmann et al, 2015) were digested with BamHI and AvrII and ligated following a standard protocol resulting in the OpIE2spl GFP1-10 vector. The OpIE2spl GFP11-MCS vector was constructed similarly, but an artificial spacer element inserted for the ease of cloning had to be removed afterwards by NheI digestion and religation.…”

Section: Vector Designmentioning

confidence: 99%

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Fast plasmid based protein expression analysis in insect cells using an automated SplitGFP screen

Bleckmann

Schmelz

Schinkowski

et al. 2016

Biotech & Bioengineering

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Recombinant protein expression often presents a bottleneck for the production of proteins for use in many areas of animal‐cell biotechnology. Difficult‐to‐express proteins require the generation of numerous expression constructs, where popular prokaryotic screening systems often fail to identify expression of multi domain or full‐length protein constructs. Post‐translational modified mammalian proteins require an alternative host system such as insect cells using the Baculovirus Expression Vector System (BEVS). Unfortunately this is time‐, labor‐, and cost‐intensive. It is clearly desirable to find an automated and miniaturized fast multi‐sample screening method for protein expression in such systems. With this in mind, in this paper a high‐throughput initial expression screening method is described using an automated Microcultivation system in conjunction with fast plasmid based transient transfection in insect cells for the efficient generation of protein constructs. The applicability of the system is demonstrated for the difficult to express Nucleotide‐binding Oligomerization Domain‐containing protein 2 (NOD2). To enable detection of proper protein expression the rather weak plasmid based expression has been improved by a sensitive inline detection system. Here we present the functionality and application of the sensitive SplitGFP (split green fluorescent protein) detection system in insect cells. The successful expression of constructs is monitored by direct measurement of the fluorescence in the BioLector Microcultivation system. Additionally, we show that the results obtained with our plasmid‐based SplitGFP protein expression screen correlate directly to the level of soluble protein produced in BEVS. In conclusion our automated SplitGFP screen outlines a sensitive, fast and reliable method reducing the time and costs required for identifying the optimal expression construct prior to large scale protein production in baculovirus infected insect cells. Biotechnol. Bioeng. 2016;113: 1975–1983. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

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Section: Resultssupporting

confidence: 89%

Section: Vector Designmentioning

confidence: 99%

Fast plasmid based protein expression analysis in insect cells using an automated SplitGFP screen

Bleckmann

Schmelz

Schinkowski

et al. 2016

Biotech & Bioengineering

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“…Production in Expi293F cells was performed using pCSE2.5-His-XP, pCSE2.6-hFc-XP or pCSE2.6-mFc-XP 64 where the respective single chain variable fragment of the antibodies or antigens were inserted by NcoI/NotI (NEB Biolabs) digestion. Antigen production in High Five insect cells was performed using NcoI/NotI compatible variants of the OpiE2 plasmid 65 containing an N-terminal signal peptide of the mouse Ig heavy chain, the respective antigen and C-terminal either 6xHis-tag, hFc or mFc.…”

Section: Design Of Expression Vectorsmentioning

confidence: 99%

SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface

Bertoglio

Meier

Langreder

et al. 2020

Preprint

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COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a novel betacoronavirus discovered in December 2019 and closely related to the SARS coronavirus (CoV). Both viruses use the human ACE2 receptor for cell entry, recognizing it with the Receptor Binding Domain (RBD) of the S1 subunit of the viral spike (S) protein.The S2 domain mediates viral fusion with the host cell membrane. Experience with SARS and MERS coronavirus has shown that potent monoclonal neutralizing antibodies against the RBD can inhibit the interaction with the virus cellular receptor (ACE2 for SARS) and block the virus cell entry. Assuming that a similar strategy would be successful against SARS-CoV-2, we used phage display to select from the human naïve universal antibody gene libraries HAL9/10 anti SARS2 spike antibodies capable of inhibiting interaction with ACE2. 309 unique fully human antibodies against S1 were identified. 17 showed more than 75% inhibition of spike binding to cells expressing ACE2, assessed by flow cytometry and several antibodies showed even an 50% inhibition at a molar ratio of the antibody to spike protein or RBD of 1:1. Furthermore, these antibodies neutralized active SARS-Cov-2 virus infection of VeroE6 cells. All 17 were all able to bind the isolated RBD, four of them with sub-nanomolar EC50. Epitope analysis of the antibodies revealed that six bind at the RBD-ACE2 interface and two on the opposite side of the domain. Universal libraries from healthy donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovered patients in a pandemic situation. 4/34 Main textIn 2015 Menachery et al. wrote: "Our work suggests a potential risk of SARS-CoV reemergence from viruses currently circulating in bat populations." 1 . Four years later, a novel coronavirus causing a severe pneumonia was discovered and later named SARS-CoV-2. The outbreak started on a sea food market in Wuhan, Hubei province (China) at the end of 2019. The disease was named COVID-19 (coronavirus disease 2019) by the World Health Organization (WHO). Sequencing showed high identity to bat corona viruses (CoV, in particular RaTG13), beta-CoV virus causing human diseases like SARS and MERS and, to a lesser extent, the seasonal CoV hCoV-OC43 and HCov-HKU1 2,3 . The spike (S) protein of SARS-CoV-2, as well as SARS-CoV, binds to the human zinc peptidase angiotensin-converting enzyme 2 (ACE2) which is expressed on lung cells, heart, kidney and intestine cells and acts as receptor for virus entry. S protein consists of the N-terminal S1 subunit, which includes the receptor binding domain (RBD), and the Cterminal S2 subunit which is anchored to the viral membrane and is required for trimerization and fusion of the virus and host membrane 4-6 . The membrane bound host protease TMPRSS2 is responsible for S protein priming by cleavage of specific sites between S1 and S2. In addition to proteolytic activation of the S2' site, conformational changes and viral entry 7-10 .Antibodies against the...

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“…Virus-free expression in insect cells presents a fast and cheap alternative for screening but is hampered by the lack of strong endogenous lepidopteran promoters. In our recent analysis of Spodoptera frugiperda promoters, we could identify promoter sequences which did not exceed activity of the strongest immediate early viral promoter [ 11 ]. Furthermore, only few Trichoplusia ni promoters are known of which the pB2-Hi5 promoter is showing the highest expression level [ 12 ].…”

Section: Introductionmentioning

confidence: 99%

“…However, the generation of stable cell lines, even using the Recombinase Mediated Cassette Exchange (RMCE) technology [ 26 ], is extremely time- and labor-intensive. Moreover, expression levels in stable cell lines are often limited for most proteins by low expression cassette copy numbers and lack of available strong constitutive promoters [ 11 ].…”

Section: Introductionmentioning

confidence: 99%

Identification of Essential Genetic Baculoviral Elements for Recombinant Protein Expression by Transactivation in Sf21 Insect Cells

et al. 2016

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The Baculovirus Expression Vector System (BEVS) is widely used to produce high amounts of recombinant proteins. Nevertheless, generating recombinant baculovirus in high quality is rather time-consuming and labor-intensive. Alternatively, virus-free expression in insect cells did not achieve similar expression levels for most proteins so far. The transactivation method is a promising approach for protein expression in Sf21 cells. It combines advantages of BEVS and plasmid-based expression by activating strong virus-dependent promoters on a transfected plasmid by baculoviral coinfection. Here, we identified expression elements required for transactivation. Therefore, we designed several vectors comprising different viral promoters or promoter combinations and tested them for eGFP expression using the automated BioLector microcultivation system. Remarkably, only the combination of the very late promoter p10 together with the homologous region 5 (hr5) could boost expression during transactivation. Other elements, like p10 alone or the late viral promoter polH, did not respond to transactivation. A new combination of hr5 and p10 with the strongest immediate early OpMNPV viral promoter OpIE2 improved the yield of eGFP by ~25% in comparison to the previous applied hr5-IE1-p10 expression cassette. Furthermore, we observed a strong influence of the transcription termination sequence and vector backbone on the level of expression. Finally, the expression levels for transactivation, BEVS and solely plasmid-based expression were compared for the marker protein eGFP, underlining the potential of transactivation for fast recombinant protein expression in Sf21 cells. In conclusion, essential elements for transactivation could be identified. The optimal elements were applied to generate an improved vector applicable in virus-free plasmid-based expression, transactivation and BEVS.

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Genomic Analysis and Isolation of RNA Polymerase II Dependent Promoters from Spodoptera frugiperda

Cited by 35 publications

References 41 publications

Fast plasmid based protein expression analysis in insect cells using an automated SplitGFP screen

Fast plasmid based protein expression analysis in insect cells using an automated SplitGFP screen

SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface

Identification of Essential Genetic Baculoviral Elements for Recombinant Protein Expression by Transactivation in Sf21 Insect Cells

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