High?￠????level expression of secreted complex glycosylated recombinant human erythropoietin in the Physcomitrella ?????￠????fuc?￠????t ?????￠????xyl?￠????t mutant

摘要

The highly glycosylated peptide hormone erythropoietin (EPO) plays a key role in the regulation of erythrocyte maturation. Currently, marketed EPO is produced by recombinant technology in mammalian cell cultures. The complementary DNA (cDNA) for human EPO (hEPO) was transiently and stably expressed in the moss Physcomitrella patens wild?￠????type and ?????￠????fuc?￠????t ?????￠????xyl?￠????t mutant, the latter containing N ?￠????glycans lacking the plant?￠????specific, core?￠????bound ???±1,3?￠????fucose and ???21,2?￠????xylose. New expression vectors were designed based on a Physcomitrella ubiquitin gene?￠????derived promoter for the expression of hEPO cDNA. Transient expression in protoplasts was much stronger at 10 than at 20???????°C. In Western blot analysis, the molecular size of moss?￠????produced recombinant human EPO (rhEPO) was identified to be 30????kDa, and it accumulated in the medium of transiently transformed protoplasts to high levels around 0.5???????μg/mL. Transgenic Physcomitrella ?????￠????fuc?￠????t ?????￠????xyl?￠????t mutant lines expressing EPO cDNA showed secretion of rhEPO through the cell wall to the culture medium. In 5?￠???? and 10?￠????L photobioreactor cultures, secreted rhEPO accumulated to high levels above 250???????μg/g dry weight of moss material after 6????days. Silver staining of rhEPO on sodium dodecylsulphate?￠????polyacrylamide gel electrophoresis (SDS?￠????PAGE) taken from the bioreactor culture demonstrated a high purity of the over?￠????expressed secreted rhEPO, with a very low background of endogenous moss proteins. Peptide mapping of rhEPO produced by the Physcomitrella ?????￠????fuc?￠????t ?????￠????xyl?￠????t mutant indicated correct processing of the plant?￠????derived signal peptide. All three N ?￠????glycosylation sites of rhEPO were occupied by complex?￠????type N ?￠????glycans completely devoid of the plant?￠????specific core sugar residues fucose and xylose.