Characterization of a Trichoplusia ni hexamerin-derived promoter in the AcMNPV baculovirus vector

López-Vidal, Javier; Gómez-Sebastián, Silvia; Sánchez‐Ramos, Ismael; Escribano, José M.

doi:10.1016/j.jbiotec.2013.03.012

Cited by 14 publications

(20 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Additionally, the Ac-ie-01 cDNA was also synthesised (GenScript) under the control of pB2 9 promoter [21]. The resulting construct ( pB2 9 Ac-ie-01 ) was cloned into the BamHI and XbaI sites of pFastBac1 .…”

Section: Methodsmentioning

confidence: 99%

Significant Productivity Improvement of the Baculovirus Expression Vector System by Engineering a Novel Expression Cassette

Gómez-Sebastián¹,

López-Vidal²,

Escribano

2014

PLoS ONE

Self Cite

View full text Add to dashboard Cite

Here we describe the development of a baculovirus vector expression cassette containing rearranged baculovirus-derived genetic regulatory elements. This newly designed expression cassette conferred significant production improvements to the baculovirus expression vector system (BEVS), including prolonged cell integrity after infection, improved protein integrity, and around 4-fold increase in recombinant protein production yields in insect cells with respect to a standard baculovirus vector. The expression cassette consisted of a cDNA encoding for the baculovirus transactivation factors IE1 and IE0, expressed under the control of the polyhedrin promoter, and a homologous repeated transcription enhancer sequence operatively cis-linked to the p10 promoter or to chimeric promoters containing p10. The prolonged cell integrity observed in cells infected by baculoviruses harbouring the novel expression cassette reduced the characteristic proteolysis and aberrant forms frequently found in baculovirus-derived recombinant proteins. The new expression cassette developed here has the potential to significantly improve the productivity of the BEVS.

show abstract

Section: Methodsmentioning

confidence: 99%

Significant Productivity Improvement of the Baculovirus Expression Vector System by Engineering a Novel Expression Cassette

Gómez-Sebastián¹,

López-Vidal²,

Escribano

2014

PLoS ONE

Self Cite

View full text Add to dashboard Cite

show abstract

“…Hence, low promoter activity might be the bottleneck. Up to today, mostly the immediate early promoter IE1 [ 10 ] combined with the enhancer element hr5 [ 10 – 12 ] as well as the Bombyx mori silkworm actin 3 promoter [ 13 ] or the Trichoplusia ni pB2 promoter [ 14 , 15 ] have been employed for stable cell line or transient plasmid based protein expression. Unfortunately, the used early baculoviral promoters are at least 10–20 times less active than the very strong late baculoviral promoters p10 and polH [ 10 , 16 ].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2015

View full text Add to dashboard Cite

The Baculoviral Expression Vector System (BEVS) is the most commonly used method for high expression of recombinant protein in insect cells. Nevertheless, expression of some target proteins-especially those entering the secretory pathway- provides a severe challenge for the baculovirus infected insect cells, due to the reorganisation of intracellular compounds upon viral infection. Therefore, alternative strategies for recombinant protein production in insect cells like transient plasmid-based expression or stable expression cell lines are becoming more popular. However, the major bottleneck of these systems is the lack of strong endogenous polymerase II dependent promoters, as the strong baculoviral p10 and polH promoters used in BEVS are only functional in presence of the viral transcription machinery during the late phase of infection. In this work we present a draft genome and a transcriptome analysis of Sf21 cells for the identification of the first known endogenous Spodoptera frugiperda promoters. Therefore, putative promoter sequences were identified and selected because of high mRNA level or in analogy to other strong promoters in other eukaryotic organism. The chosen endogenous Sf21 promoters were compared to early viral promoters for their efficiency to trigger eGFP expression using transient plasmid based transfection in a BioLector Microfermentation system. Furthermore, promoter activity was not only shown in Sf21 cells but also in Hi5 cells. The novel endogenous Sf21 promoters were ranked according to their activity and expand the small pool of available promoters for stable insect cell line development and transient plasmid expression in insect cells. The best promoter was used to improve plasmid based transient transfection in insect cells substantially.

show abstract

“…Thiem & Miller (Thiem and Miller 1990) showed that the combination of the vp39 and the polyhedrin promoter enhanced the expression of foreign genes compared to using those promoters alone in Sf cells, because this hybrid promoter showed regulation patterns of late and very late promoters. López-Vidal et al (López-Vidal et al 2013) also demonstrated an increase in GFP production of more than 20% at early times post-infection, and similar expression levels at very late times postinfection in Sf21 cells using a pB2-p10 promoter combination, with respect to conventional late promoters.…”

Section: Discussionmentioning

confidence: 65%

“…Other strategies were based on the deletion of non-essential genes of the vector (Hitchman et al 2010;Hitchman et al 2011). One standard strategy is the search for promoters which are stronger than those commonly used, such as the p10 and polyhedrin (pph) promoters, or chimeras of them employed in laboratory and industrial production (Thiem and Miller 1990;Ishiyama and Ikeda 2010;Lin and Jarvis 2012;López-Vidal et al 2013). However, often the efficiency of the promoter also depends on the regulatory sequences around them and the type of cellular lines in which they are acting (Matsuura et al 1987;Morris and Miller 1992;C H Gross and Rohrmann 1993;Lo et al 2002).…”

Section: Discussionmentioning

confidence: 99%

“…To date, different types of promoters have been tested in the BEVS to improve recombinant protein expression. Viral promoters such as vp39 or 39K, and promoters derived from insect larvae such as the hexamerinderived promoter pB2 from Trichoplusia ni (López-Vidal et al 2013) showed high levels of expression of recombinant proteins. In other cases, the combination of some of these promoters with the conventional promoters exhibited higher expression levels of the recombinant proteins than the standard late promoters alone (Thiem and Miller 1990;Morris and Miller 1992;Ishiyama and Ikeda 2010;Lin and Jarvis 2012).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Untitled

Supplemental Information 3: Figure 5 Values

View full text Add to dashboard Cite

The baculovirus expression vector system (BEVS) has been widely used to produce a large number of recombinant proteins, and is becoming one of the most powerful, robust, and cost-effective systems for the production of eukaryotic proteins. Nevertheless, as in any other protein expression system, it is important to improve the production capabilities of this vector. The orf46 viral gene was identified among the most highly abundant sequences in the transcriptome of Spodoptera exigua larvae infected with its native baculovirus, the S. exigua multiple nucleopolyhedrovirus (SeMNPV). Different sequences upstream of the orf46 gene were cloned, and their promoter activities were tested by the expression of the GFP reporter gene using the Autographa californica nucleopolyhedrovirus (AcMNPV) vector system in different insect cell lines (Sf21, Se301, and Hi5) and in larvae from S. exigua and Trichoplusia ni. The strongest promoter activity was defined by a 120 nt sequence upstream of the ATG start codon for the orf46 gene. On average, GFP expression under this new promoter was more than two fold higher than the expression obtained with the standard polyhedrin (pph) promoter. Additionally, the orf46 promoter was also tested in combination with the pph promoter, revealing an additive effect over the pph promoter activity. In conclusion, this new characterized promoter represents an excellent alternative to the most commonly used baculovirus promoters for the efficient expression of recombinant proteins using the BEVS.

show abstract

Characterization of a Trichoplusia ni hexamerin-derived promoter in the AcMNPV baculovirus vector

Cited by 14 publications

References 32 publications

Significant Productivity Improvement of the Baculovirus Expression Vector System by Engineering a Novel Expression Cassette

Significant Productivity Improvement of the Baculovirus Expression Vector System by Engineering a Novel Expression Cassette

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

Untitled

Contact Info

Product

Resources

About