Poster
Presentation 1B-12
HEX1
as a Vehicle for Foreign Protein Expression in Filamentous Fungi such as Trichoderma reesei
P. L. Bergquist,1,2* N. C.
Curach,1 V. S. J. Te’o,1 M. D. Gibbs1 and K.
M. H. Nevalainen1
1Department of Biological Sciences
Biotechnology
Research Institute
Phone: +61 2 9850 8614
Fax: +61 2 9850 9748
Email: peter.bergquist@mq.edu.au
2Department of Molecular Medicine &
Pathology,
Strong promoters that
function under specific conditions are required for effective gene expression
of industrially-important gene products. We are investigating the catabolite repression-insensitive
hex1 gene promoter as an alternative
to inducible gene promoters in Trichoderma
reesei. A 4376 bp DNA fragment that
contains the hex1 gene open reading
frame (ORF) as well as promoter and terminator sequences was isolated from T. reesei using a Genomic Walking PCR
procedure. The 784 bp hex1 gene ORF gives a peptide sequence
of 225 amino acids with an expected molecular mass of 25,207 when
translated. HEX1 isolated from the cell
envelope fraction of T. reesei, is
expressed in different iso-forms also seen on 2-D gels as described
earlier. Multiple
transcription start sites (tsp) and
the 5’ untranslated region (UTR) were identified by 5’RACE RT-PCR. There are three hex1 transcript types, two of which originate from two tsps at approximately –320 and –1335
from the start codon, and are separated by a 500 bp intron within the
5’UTR. The third transcript type results
from alternative splicing of the intron within the coding sequence at the 3’
end, which results in the inclusion or exclusion of an unconserved
histidine-rich coding region at the HEX1 N-terminus. Northern blot results showed that high levels
of hex1 mRNA were expressed during
the first 24 hrs of growth in cellulase-inducing and non-inducing medium, which
could be beneficial for the expression of foreign proteins for industrial
applications.