Expression of granulocyte colony stimulating factor (GCSF) in Hansenula polymorpha.
OBJECTIVE
During past decades Hansenula polymorpha has attracted global attention for the expression of recombinant proteins due to its high growth rate, minimal nutritional porequirements and use of methanol as a low cost inducer.


METHODS
The corresponding nucleotide sequences for the expression of heterologous genes in Hansenula poylmorpha were extracted and assembled in an E. coli vector. The constructed expression cassette included formate dehydrogenase promoter (pFMD), a secretory signal sequence, a multiple cloning site (MCS) and methanol oxidase (MOX) terminator. Zeocin resistance gene fragment and complete cDNA encoding granulocyte colony stimulating factor (GCSF) were cloned downstream of the expression cassette in-frame with signal sequence. Restriction mapping and sequence analysis confirmed the correct cloning procedures. Final vector was transformed into Hansenula and recombinant host was induced for the expression of GCSF protein by adding methanol. SDS-PAGE and immuno-blotting were performed to confirm the identity of r-GCSF.


RESULTS
The expression cassette containing gcsf gene (615bp) and zeocin resistance marker (sh-ble, 1200bp) was prepared and successfully transformed into competent Hansenula polymorpha cells via electroporation. Zeocin resistant colonies were selected and GCSF expression was induced in recombinant Hansenula transformants using 0.5% methanol and an approximately 19kDa protein was observed on SDS-PAGE. Western blot analysis using serum isolated from GCSF-treated rabbit confirmed the identity of the protein.


CONCLUSIONS
Molecular studies confirmed the designed expression cassette containing gcsf gene along with pFMD and signal sequence. The expressed 19kDa protein also confirmed the ability of designed vector in expressing heterologous genes in Hansenula cells.
Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
Yeganeh
Y
Talebkhan
Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
Tannaz
T
Samadi
Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran; Department of Biology, Azad University of Damghan, Iran.
Armin
A
Samie
Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
Farzaneh
F
Barkhordari
Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
Mohammad
M
Azizi
Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
Vahid
V
Khalaj
Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
Esmat
E
Mirabzadeh
Expression cassette; GCSF; Hansenula polymorpha