In vivo de-glycosylation of recombinant proteins by co-expression with PNGase F
Inventors
Mammedov, Tarlan • Yusibov, Vidadi
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11673926-B2
Publication Date
2023-06-13
Expiration Date
Abstract
Materials and methods for in vivo de-glycosylation of recombinant N-glycosylated proteins by co-expression with bacterial PNGase F (Peptide: N-glycosidase F) in plants, using a transient expression system are described. Methods are described which, for example, produce recombinant proteins of interest in plants in a non-glycosylated form. A method of expressing active bacterial PNGase F in plants also is provided.
Core Innovation
The invention relates to generating a deglycosylated polypeptide of interest in a plant by transiently expressing a bacterial PNGase F polypeptide fusion protein in plant cells and transiently expressing the polypeptide of interest after infiltration. The bacterial PNGase F polypeptide is provided as a fusion with a plant signal peptide and is transiently expressed by activation of a promoter. The polypeptide of interest includes a glycosylation site and is transiently expressed in vivo by infiltration.
The bacterial PNGase F and the polypeptide of interest include an endoplasmic reticulum retention sequence. By action of the PNGase F polypeptide, all N-linked glycans of the polypeptide of interest are cleaved in vivo. The disclosure additionally includes an engineered bacterial PNGase F fusion comprising a plant signal peptide, endoplasmic reticulum retention (KDEL), and a purification tag.
The described approach produces uniformly N-deglycosylated recombinant proteins in plants via transient co-expression of an active bacterial PNGase F in plant cells. In the disclosed examples, transient expression is performed in Nicotiana benthamiana using glycosylated polypeptides including a malaria vaccine candidate Pfs48F1, Bacillus anthracis protective antigen, and an anti-PA antibody. In vivo-deglycosylated Pfs48F1 shows improved antibody recognition compared with glycosylated and in vitro-deglycosylated forms, and a comparison to chloroplast-targeted expression indicates weaker antibody binding.
Claims Coverage
The provided independent claim contains the core inventive concept of in vivo, complete N-deglycosylation of a plant-expressed target polypeptide by transiently co-expressing a bacterial PNGase F fusion targeted to the endoplasmic reticulum. The dependent claims further refine delivery/construct configuration, host plant cell choice, sequence identity constraints for PNGase F, and narrowing the target polypeptide type.
Plant transient expression of bacterial PNGase F fusion
Introducing into a plant cell a first nucleic acid comprising a nucleotide sequence encoding a bacterial PNGase F polypeptide fusion protein comprising a plant signal peptide, operably linked to a promoter such that the bacterial PNGase F polypeptide is transiently expressed by infiltration.
Transient expression of a glycosylated polypeptide of interest in plant
Introducing into the plant cell a second nucleic acid comprising a second nucleotide sequence encoding the polypeptide of interest, operably linked to a second promoter such that the polypeptide of interest is expressed by activation of the second promoter, wherein the nucleotide sequence includes a sequence not naturally found in the plant cell, wherein the polypeptide contains a glycosylation site, and wherein the polypeptide of interest is transiently expressed by infiltration.
Endoplasmic reticulum retention for PNGase F and target
The PNGase F polypeptide and the polypeptide of interest include an endoplasmic reticulum retention sequence.
In vivo cleavage of all N-linked glycans
By action of the PNGase F polypeptide, cleaves all N-linked glycans of the polypeptide of interest in vivo.
Single nucleic acid construct for both expression elements
The first and second nucleic acids are included together in a single nucleic acid construct.
Nicotiana benthamiana plant cell host
The method is performed using a Nicotiana benthamiana plant cell.
PNGase F nucleotide sequence identity constraint
The first nucleotide sequence is SEQ ID NO: 1.
Agrobacterium construct delivery of both nucleic acids
Introducing two nucleic acids into a plant cell using an Agrobacterium construct.
Target polypeptide as a pharmaceutical protein
The polypeptide of interest is a pharmaceutical protein.
Overall, the claim coverage centers on producing a deglycosylated polypeptide in a plant by transient co-expression of a bacterial PNGase F fusion targeted to the endoplasmic reticulum and a glycosylated polypeptide of interest, followed by in vivo cleavage of all N-linked glycans; dependent claims narrow the configuration, host, PNGase F sequence identity, delivery format, and target protein type.
Stated Advantages
Produces uniformly N-deglycosylated recombinant proteins in plants.
Improves antibody recognition for in vivo-deglycosylated Pfs48F1 versus glycosylated and in vitro-deglycosylated forms.
Enables cleavage of all N-linked glycans of the polypeptide of interest in vivo.
Documented Applications
Generating deglycosylated forms of a malaria vaccine candidate Pfs48F1 for improved antibody recognition.
Generating deglycosylated forms related to Bacillus anthracis protective antigen and an anti-PA antibody.
Comparing the disclosed ER-targeted PNGase F approach to chloroplast-targeted expression (Arm-Pfs48F1) in terms of antibody binding.
Interested in licensing this patent?