Production of recombinant human erythropoietin from Pichia pastoris and its structural analysis.
OBJECTIVE
To design and investigate a recombinant expression system producing a therapeutically important glycoprotein, human erythropoietin (rHuEPO), by Pichia pastoris.


RESULTS
EPO cDNA was cloned into pPICZalphaA for expression under control of AOX1 promoter and fused, on the amino-terminal end, with a polyhistidine tag for rapid purification. A target site for factor Xa protease was also introduced, such that cleavage in vitro produced a mature form of rHuEPO having the native N- and C-termini. RHuEPO was characterized as to the extent and nature of N-linked glycosylation using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and western blotting. The rHuEPO produced was approximately 30 kDa. All three N-linked glycosylation sites were occupied dominantly by Man(17)(GlcNAc)(2). N-glycanase-treated rHuEPO purified but not digested with factor-Xa-protease, showed a spectral peak centered about m/z 20400 Da.


CONCLUSIONS
The native polypeptide form of human EPO (c. 18 kDa) was obtained for the first time in P. pastoris expression system, after affinity purification, deglycosylation and factor-Xa-protease digestion. The amount of sodium dodecyl sulfate used prior to deglycosylation was found to be crucial in determining the dominant form of glycan in glycoproteins.


CONCLUSIONS
The novel approaches to protein expression and purification system and structural analysis presented, would be important especially for therapeutic proteins expressed in P. pastoris.
Chemical Engineering Department, Industrial Biotechnology and Metabolic Engineering Laboratory, Middle East Technical University, Ankara, Turkey.
E
E
Celik
P
P
Calik
S M
SM
Halloran
S G
SG
Oliver
D000595:Amino Acid Sequence; D019149:Bioreactors / Q000382:microbiology*; D015153:Blotting, Western / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection / Q000379:methods
D011994:Recombinant Proteins; D004921:Erythropoietin