Recombinant proteins are deglycosylated in vivo by co-expression with PNGase F
A deglycosylation and expression system technology, applied in the field of deglycosylation of recombinant proteins in vivo through co-expression with PNGase F, can solve problems such as satisfactory realization
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[0111] Example 1 - Materials and methods
[0112] Cloning and expression of PNGase F in Nicotiana benthamiana: The PNGase F gene was optimized for expression in Nicotiana benthamiana (for codon optimization, mRNA stability, RNA unstable sequence knockout, etc.) Synthesized at GENEART AG (Regensburg, Germany) with sites flanking PacI (5'-end) and XhoI (3'-end, after the stop codon). To express PNGaseF in N. benthamiana plants using a transient expression system, the nucleotides encoding the signal peptide (amino acids 1-40) were removed from the PNGase F sequence and the signal peptide encoding the tobacco PR-1a (MGFVLFSQLPSFLLVSTLLLFLVISHSCRA; SEQ ID NO: Nucleotides of NO: 3) are added to the 5' end of the coding sequence. Sequences encoding an ER resident signal (KDEL; SEQ ID NO: 5) and a FLAG epitope affinity purification tag (SEQ ID NO: 4) were added to the 3' end. The resulting sequence was inserted into the delivery vector pGRD4 (Roy et al. (2010) Virol. 405(1):93-99)...
example 2
[0129] Example 2 - Expression of bacterial PNGase F in Nicotiana benthamiana
[0130] The PNGase F coding sequence (314 amino acids, representing the full-length catalytically active protein without a signal sequence) was optimized as described in Example 1 (Figure 1), cloned into the pGRD4 expression vector, and grown in N. benthamiana in the expression. The average expression level of PNGase F was about 150 mg / kg fresh biomass. Expression of PNGase F was confirmed by immunoblot analysis using an anti-FLAG monoclonal antibody (see Figure 2A ).
[0131] Example 3 - Co-expression of Pfs48F1 with bacterial PNGase F
[0132] The Pfs48F1E malaria vaccine candidate was transiently co-expressed with bacterial PNGase F in N. benthamiana to test whether N-linked oligosaccharides would be cleaved from Pfs48F1 in an in vivo setting. PNGase F was expressed with the FLAG epitope followed by the C-terminal ER resident signal KDEL (SEQ ID NO: 5). The results are presented in Figur...
example 4
[0133] Example 4 - Protective Antigen (PA) of Bacillus anthracis and Antigen (C) against PA of Bacillus anthracis Coexpression of PNGase F
[0134] The in vivo deglycosylation strategy was applied to two other glycoproteins, the B. anthracis protective antigen (PA) and antibodies against B. anthracis PA. The co-expression results are presented in FIG. 4 . A mobility shift was observed in the heavy chain (HC) with one glycosylation site, but not in the light chain (LC) lacking the glycosylation site. These results, together with coexpression with Pfs48F1, indicate that PNGase F successfully cleaves N-linked glycans from all glycoproteins tested, and that this strategy can be used to produce therapeutic agents in deglycosylated form using a transient expression system in N. benthamiana. proteins and antibodies.
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