Cloning And Expression Of Synthetic Human Erythropoietin In Methylotrophic Yeast, Pichia Pastoris
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Date
2010-11
Authors
Bustami, Yazmin
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Sains Malaysia
Abstract
Human erythropoietin is a hormone or glycoprotein produced by the kidney
and bone marrow with important physiological functions, such as, erythropoiesis,
angiogenesis, and wound healing. The natural source of huEpo is purified from
human urine (uhuEPO). Alternatively, recombinant human erythropoietin could
overcome the limited supply of uhuEPO. Thus, the focus of this study is to generate a
synthetic huEPO gene and express in Pichia pastoris. PCR assembly method utilized
to construct a synthetic gene using twenty sets of overlapping oligonucleotides,
covering the huEPO A gene sequence and two newly introduced restriction enzyme
sites. Results from DNA sequence analysis showed accurate assembled synthetic
huEPO gene albeit minor base mutations detected. A free-mutation synthetic huEPO
A gene successfully obtained using site directed mutagenesis system. Then it was
sub-cloned into pPIC9k and was transformed into Pichia strain, GS115.
Transformation of the linearized recombinant construct successfully integrated at the
HIS4 locus between the recombinant plasmid and the P. pastoris genome, hence,
generating His+ Mut+ transformants. The small scale expression studies were carried
out and the cultures were induced with methanol for three days (0 to 72 hr) with 24
hr interval time. Secreted rhuEPO protein was observed at approximately 30 kDa
band and further confirmed using ELISA analysis. Based on ELISA analysis, the
highest amount of Epo concentration was observed at 72 hr.
Description
Keywords
Pichia pastoris , human erythropoietin