74NISR RESEARCH GRANT
2004 Research Grant

Development of Efficient Recombinant Protein Expression
System via mRNA Export Mechanism
Seiji Masuda
Graduate School of Biostudies, Kyoto University

Research aims
The proteins that is modified with sugar chain for its invivo activity like drugs are essential to be produced inmammalian cells. Protein production in mammalian cells,
however, is much less efficient production compared within bacteria such as E. coli. The use of virus mRNA containing CTE can bypass the cellular mRNA pathway without
splicing and the transfer of this virus export pathway tomammalian protein production will improve the protein
production than ever tried. Recently, the mechanism whymRNA with CTE is independent of splicing was found.1
CTE binds specifically to mRNA export cargo, Tap/p15
heterodimer, thereby mRNA containing CTE does not needsplicing for its export. The method used in this projectis expected to achieve a highly efficient protein production in mammalian cells because this method enhances theexport pathway and it is different from promoter enhancement in cell. The final goal of this project is to make theSUPERCELL for the protein production in mammaliancells.
Methods
Luciferase expression plasmid containing CTE(plucCTE) was a gift from Dr. Kimura T.2 CTE was removed
from plucCTE to make control plasmid(pluc).
pCMVHATap was from Dr. Ferber, BK.3 pCMVflagp15
was constructed by using p15 full length PCR product amplified from HeLa cDNA library. The pcDNA5/FRT/HATap
was constructed by inserting HATap into pcDNA5/FRT.
DNA was transfected into 293T cells using lipofectamine2000. Luciferase activity was measured with luminometer. 293T cells were cultured as standard method.
HATap was detected with western analysis.
ResultsThe analysis of a novel expression system was done bymeasuring luciferase activity. Luciferase expression in thepresence of CTE enhanced its activity by 30% than in theabsence of CTE. Its enhancement, however, was not as expected. It is suggested that transient expression of large
amount of mRNA may cause the lack of mRNA export
cargo, Tap/p15, thereby the luciferase expressed in the nucleus was not export efficiently. To solve the lack of export

receptor, inducible expression of Tap/p15 was examined.
First, Ha tagged Tap was inserted into pcDNA5/FRT vector
to make pcDNA5/FRT/HATap whose expression was induced by Tet and/.or Dox. pcDNA5/FRT/HATap was introduced into 293Trex cells with pOG44 which express Frp
recombinase to make 293HATap cell. 293Trex cell can integrates 1 copy of target DNA into its genome. As a result,
HATap expression was very similar between 15 clones obtained. Second, using one of these clones (No1), flag tagged
p15 was introduced and now the best clone is selecting
using flag antibody.
Conclusion
It becomes obvious that the validity of CTE mediatedmRNA export system to produce protein whose activity isdependent of mammalian cell production, may be difficultfrom the data of transient expression trial in the absence ofexogenous expression of Tap/p15. Then, the way to better
estimation is to make a clone that express Tap/p15 export
cargo proteins. After obtaining the clone, validity of CTE
mediated mRNA export system will bwe examined. CTE.
The position of CTE, then, will be examined to obtain theinformation where CTE is the best for the expression. It is
not described in this report, there is another method to enhance the protein production using the export system. Thisuses the export pathway in its cell. The TREX complex is
responsible for the global mRNA export pathway,4,5 then if
the expression of TREX was well tuned, the mRNA export
pathway will be activated than the standard condition.
TREX consists of 8 proteins. The complete information ofTREX takes 3 years of time.5 The tight regulation of these
TREX complex protein expression, then, may have a great
difficulty to set the efficient protein production system up ina short period. The best way to develop the efficient protein
production system using the mRNA export patway is focusing on the construction of virus CTE system and looking
for the production rate by changing the condition ofTap/p15 expression.
References
1. Gruter, P., et al., TAP, the human homolog of Mex67p, mediates
CTE-dependent RNA export from the nucleus. Mol. Cell, 1, 649–659,
1998.
2. Kimura, T., et al., CRM1-dependent, but not ARE-mediated, nuclear export of IFN-a 1 mRNA. J. Cell. Sci., 117, 2259–2270.

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3. Bear, J., et al., Identification of novel import and export signals ofhuman Tap, the protein that bind to constitutive transport element of thetype D retrovirus mRNAs. Mol. Biol. Chem., 19, 6306–6317, 1999.
4. Stresser, K., et al., TREX is a conserved complex coupling transcription

with messenger RNA export. Nature, 417, 304–308, 2002.
5. Masuda, S., et al., Recruitment of the human TREX complex to mRNAduring splicing. Genes Dev., 19, 1512–1517, 2005.