توليد فاكتور 9 انعقادي انساني هايپرگليكوزيله در سلول هاي جنيني كليه انسان (HEK293) با رويكرد مهندسي گليكوزيلاسيون

Production of a Novel Hyper-Glycosylated Human Coagulation Factor IX in Human Embryonic Kidney 293 Cells (HEK293T Cells), Using a Glyco-Engineering Approach

ﻫﺎﯾﭙﺮﮔﻠﯿﮑﻮزﯾﻼﺳﯿﻮن ﻓﺮآﯾﻨﺪي اﺳﺖ ﮐﻪ در آن ﺑﺎ اﺳﺘﻔﺎده از روشﻫﺎي ﻣﻬﻨﺪﺳﯽ ژﻧﺘﯿﮏ و ﺟﻬﺶزاﯾﯽ ﻫﺪﻓﻤﻨﺪ ﯾﮏ ﺟﺎﯾﮕﺎه ﺟﺪﯾﺪ N- ﮔﻠﯿﮑﻮزﯾﻼﺳﯿﻮن )ﻣﻮﺗﯿﻒ Asn-Xxx-Ser/Thr( ﺑﻪ درون ﺗﻮاﻟﯽ اﺳﯿﺪآﻣﯿﻨﻪاي ﭘﺮوﺗﺌﯿﻦ ﻧﻮﺗﺮﮐﯿﺐ اﺿﺎﻓﻪ ﻣﯽﺷﻮد. در ﺻﻮرت اﺗﺼﺎل ﮔﻠﯿﮑﺎن ﺟﺪﯾﺪ ﺑﻪ آﺳﭙﺎراژﯾﻦ ﻣﻮﺟﻮد در اﯾﻦ ﻣﻮﺗﯿﻒ، ﻫﺎﯾﭙﺮﮔﻠﯿﮑﻮزﯾﻼﺳﯿﻮن رخ ﻣﯽدﻫﺪ. اﯾﻦ ﻓﺮآﯾﻨﺪ ﺑﻪوﯾﮋه در ﺻﻨﻌﺖ ﺗﻮﻟﯿﺪ ﭘﺮوﺗﺌﯿﻦﻫﺎي داروﯾﯽ ﺑﺴﯿﺎر ﻣﻮرد ﺗﻮﺟﻪ اﺳﺖ. وﯾﮋﮔﯽﻫﺎي ﻓﺎرﻣﺎﮐﻮﮐﯿﻨﺘﯿﮑﯽ داروﻫﺎي ﻧﻮﺗﺮﮐﯿﺐ، ﻣﻤﮑﻦ اﺳﺖ ﺗﺤﺖ ﺗﺎﺛﯿﺮ ﮔﻠﯿﮑﺎن اﺿﺎﻓﻪﺷﺪه ﻗﺮار ﮔﯿﺮد و ﻧﯿﻤﻪ ﻋﻤﺮ آﻧﻬﺎ اﻓﺰاﯾﺶ ﯾﺎﺑﺪ. در ﻣﻄﺎﻟﻌﻪ ﺣﺎﺿﺮ، ﺑﺮ اﺳﺎس ﻣﻄﺎﻟﻌﺎت ﺑﯿﻮاﻧﻔﻮرﻣﺎﺗﯿﮑﯽ، در داﻣﻨﻪ ﮔﻼي ﻓﺎﮐﺘﻮر 9 اﻧﺴﺎﻧﯽ ﯾﮏ ﺟﺎﯾﮕﺎه ﺟﺪﯾﺪ N- ﮔﻠﯿﮑﻮزﯾﻼﺳﯿﻮن از ﻃﺮﯾﻖ ﺟﻬﺶزاﯾﯽ ﻫﺪﻓﻤﻨﺪ ﻣﺒﺘﻨﯽ ﺑﺮ PCR و ﺗﺒﺪﯾﻞ ﮐﺪون اﺳﯿﺪآﻣﯿﻨﻪ آرژﯾﻨﯿﻦ ﺷﻤﺎره 37 ﺑﻪ آﺳﭙﺎراژﯾﻦ اﯾﺠﺎد ﺷﺪ. ﺗﻮاﻟﯽ رﻣﺰﮐﻨﻨﺪه ﻓﺮم ﺟﻬﺶﯾﺎﻓﺘﻪ ﻓﺎﮐﺘﻮر 9 اﻧﺴﺎﻧﯽ ﺑﻪﻃﻮر ﻣﻮازي ﺑﺎ ﺗﻮاﻟﯽ ﻃﺒﯿﻌﯽ )ﺑﻪﻋﻨﻮان ﮐﻨﺘﺮل(، در وﮐﺘﻮر ﺑﯿﺎﻧﯽ pCEP4 ﻣﺠﻬﺰ ﺑﻪ ﭘﺮوﻣﻮﺗﺮ وﯾﺮوس ﺳﺎﯾﺘﻮﻣﮕﺎﻟﻮ (CMV)، ﻫﻤﺴﺎﻧﻪﺳﺎزي ﺷﺪ و ﺑﯿﺎن آﻧﻬﺎ در ﺳﻠﻮلﻫﺎي HEK293T ﻣﻮرد ﺑﺮرﺳﯽ ﻗﺮار ﮔﺮﻓﺖ. ﺑﻪﻣﻨﻈﻮر ﺑﺮرﺳﯽ رﺧﺪاد ﻫﺎﯾﭙﺮﮔﻠﯿﮑﻮزﯾﻼﺳﯿﻮن در ﻧﻮع ﺟﻬﺶﯾﺎﻓﺘﻪ، از روش ﮔﺮادﯾﺎن SDS-PAGE داراي ﺷﯿﺐ ﻏﻠﻈﺖ آﮐﺮﯾﻞآﻣﯿﺪ و ﺑﻪ دﻧﺒﺎل آن وﺳﺘﺮنﺑﻼﺗﯿﻨﮓ اﺳﺘﻔﺎده ﺷﺪ ﮐﻪ ﻧﺸﺎندﻫﻨﺪه ﺳﻨﮕﯿﻦﺗﺮﺑﻮدن ﻣﺤﺼﻮل ﺟﻬﺶﯾﺎﻓﺘﻪ ﻧﺴﺒﺖ ﺑﻪ ﻓﺮم ﻃﺒﯿﻌﯽ ﺑﻮد. ﻫﻤﭽﻨﯿﻦ ﭘﺲ از ﻫﻀﻢ ﺑﺎ اﺳﺘﻔﺎده از آﻧﺰﯾﻢ PNGase، ﻫﺮ دو ﻣﺤﺼﻮل ﻃﺒﯿﻌﯽ و ﺟﻬﺶﯾﺎﻓﺘﻪ وزن ﻣﻮﻟﮑﻮﻟﯽ ﯾﮑﺴﺎﻧﯽ ﮐﺴﺐ ﮐﺮدﻧﺪ. اﯾﻦ ﻧﺘﺎﯾﺞ ﻧﺸﺎن داد ﮐﻪ اﻓﺰاﯾﺶ وزن ﻣﻮﻟﮑﻮﻟﯽ ﭘﺮوﺗﺌﯿﻦ ﺟﻬﺶﯾﺎﻓﺘﻪ ﻧﺎﺷﯽ از اﺿﺎﻓﻪﺷﺪن ﮔﻠﯿﮑﺎن ﺟﺪﯾﺪ ﺑﻮده و ﺗﺎﯾﯿﺪﮐﻨﻨﺪه رﺧﺪاد ﻫﺎﯾﭙﺮﮔﻠﯿﮑﻮزﯾﻼﺳﯿﻮن اﺳﺖ.

Hyper-glycosylation is an approach to introduce new N-glycosylation consensus sequence (s) (Asn-Xxx-Ser/Thr three-peptide) into a protein primary amino acid sequences by site-directed mutagenesis which is followed by the attachment of a new glycan to the Asn residue located within the three-peptide sequence. Hyper-glycosylation has attracted lots of interest especially in the protein therapeutics industry. The attached glycan may improve the pharmacokinetic properties of the hyper-glycosylated proteins and increase their half-life in the bloodstream. In the current study, a new N-glycosylation site was introduced into N-terminal Gla domain of hFIX. Arg37 position of mature hFIX was targeted to be converted into Asn residue by site-directed mutagenesis using overlap extension PCR. Recombinant expression plasmids for native and mutant hFIX were constructed. The expression of the recombinant wild-type and mutant hFIX was analyzed in mammalian HEK293T cells using gradient SDS-PAGE and western blotting analysis. The results indicated in higher molecular weight for R37N mutant in compared with the native protein. The glycan attachment to R37N mutant was further confirmed by PNGase digestion and western blotting