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Effect of promoter
strength and signal sequence on the periplasmic expression
of human interferon-α2b in Escherichia coli
Ramakrishnan Nagasundara Ramanan1,
Wong Bee Tik1, Hamid Rajabi Memari1,2,
Siti Nor Ani Azaman1, Tau Chuan Ling3,
Beng Ti Tey1,4, Mohd. Azmi Mohd. Lila1,
Mohd. Puad Abdullah5, Raha Abdul Rahim1,5
and Arbakariya B. Ariff1,6*
1Institute
of Bioscience, Universiti Putra Malaysia, 43400 Serdang,
Selangor, Malaysia.
2Shahid
Chamran University, Golestan BLV. Ahvaz, Iran.
3Department
of Process and Food Engineering, Faculty of Engineering,
Universiti Putra Malaysia, 43400 UPM Serdang, Selangor,
Malaysia.
4Department
of Chemical and Environmental Engineering, Faculty
Engineering, Universiti Putra Malaysia, 43400 UPM Serdang,
Selangor, Malaysia.
5Department
of Cell and Molecular Biology, Faculty of Biotechnology and
Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM
Serdang, Selangor, Malaysia.
6Department
of Bioprocess Technology, Faculty of Biotechnology and
Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM
Serdang, Selangor, Malaysia.
*Corresponding author. E-mail:
arbarif@biotech.upm.edu.my.
Tel: +603-8946 7591. Fax: + 603-8946 7593.
Abbreviation: ompA,
outer membrane protein A; pelB, pectate lyase B;
PCR, Polymerase chain reaction;
ATCC,
American type culture collection; RNA, Ribonucleic
acid; rRNA, ribosomal RNA; RBS, Ribosomal
binding site
Accepted
21 August, 2009 |
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Two plasmids, pFLAG-ATS and pET 26b(+), were studied for the
periplasmic expression of recombinant human interferon-α2b
(IFN-α2b) in Escherichia coli. The pFLAG-ATS contains
ompA signal sequence and tac promoter while pET
26b(+) contains pelB signal sequence and T7lac
promoter. It was observed that periplasmic expression of
IFN-α2b from pET 26b(+) was around 3000 times higher than
pFLAG-ATS. Difference in the expression level was attributed
to the difference in the promoters and the signal sequences.
In
silico
analysis of mRNA secondary structures were analyzed using
Vienna RNA package and MFOLD. The results
suggested that the
increase of expression would mainly due to the difference in
the translation initiation associated with secondary
structure of mRNA transcribed by both
plasmids.
Key
words:
Interferon-α2b, Periplasm, Signal sequence, mRNA secondary
structure, Escherichia coli. |
