Genomic Analysis and Isolation of RNA Polymerase II Dependent Promoters from Spodoptera frugiperda.
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.
Department of Structure and Function of Proteins, Research Group Recombinant Protein Expression, Helmholtz Centre for Infection Research, Braunschweig, Germany.
Maren
M
Bleckmann
Department of Protein Expression and Purification Core Facility & Genomics Core Facility, EMBL Heidelberg, Germany.
Markus H-Y
MH
Fritz
Department of Genome Analytics, Helmholtz Centre for Infection Research, Braunschweig, Germany.
Sabin
S
Bhuju
Department of Genome Analytics, Helmholtz Centre for Infection Research, Braunschweig, Germany.
Michael
M
Jarek
Department of Structure and Function of Proteins, Research Group Recombinant Protein Expression, Helmholtz Centre for Infection Research, Braunschweig, Germany.
Margitta
M
Schürig
Department of Genome Analytics, Helmholtz Centre for Infection Research, Braunschweig, Germany.
Robert
R
Geffers
Department of Protein Expression and Purification Core Facility & Genomics Core Facility, EMBL Heidelberg, Germany.
Vladimir
V
Benes
Department of Protein Expression and Purification Core Facility & Genomics Core Facility, EMBL Heidelberg, Germany.
Hüseyin
H
Besir
Department of Structure and Function of Proteins, Research Group Recombinant Protein Expression, Helmholtz Centre for Infection Research, Braunschweig, Germany.
Joop
J
van den Heuvel
D000818:Animals; D002460:Cell Line; D015870:Gene Expression; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D023281:Genomics / Q000379:methods; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D018411:Spodoptera / Q000235:genetics* / Q000378:metabolism*; D059467:Transcriptome
D012333:RNA, Messenger; D012319:RNA Polymerase II