Cloned (Comment) | Organism |
---|---|
functional recombinant expression of His6-tagged human furin lacking its signal peptide in Nicotiana benthamiana plants. Plant-produced human furin is highly active both in vivo and in vitro and specifically cleaves the tested target proteins, factor IX (FIX) and protective antigen (PA83). Plants lack some of the important posttranslational modifications, including furin processing, but both, enzymatic deglycosylation and proteolytic processing of target proteins, can be achieved in vivo by coexpression of deglycosylating and furin cleavage enzymes in a single cell to produce deglycosylated and furin processed target proteins. Recombinant coexpression of engineered enzyme and substrate using the Agrobacterium tumefaciens strain AGL1 transfection method, expression in leaves. Coexpression of human furin with PA83 of Bacillus anthracis and deglycosylation enzymes Endo H or PNGase F in Nicotiana benthamiana. Deglycosylation efficiency of plant produced Endo H is greater than that of plant produced PNGase F. When full length furin is coexpressed with the PA83 protein in Nicotiana benthamina plants, there is little or no cleavage of PA83. Plant-produced human furin treated with PNGase F and Endo H does not exhibit cleavage activity | Homo sapiens |
Protein Variants | Comment | Organism |
---|---|---|
additional information | engineering of the human furin gene for expression in plants, the production of a functional active recombinant truncated human furin in Nicotiana benthamiana plants. The signal peptide (amino acids 1-25) is removed from the furin sequence, and Nicotiana tabacum PR-1a signal peptide (MGFVLFSQLPSFLLVSTLLLFLVISHSCRA) is added to the N-terminus. The KDEL sequence (the endoplasmic reticulum retention signal) and the His6 tag (the affinity purification tag) are added to the C-terminus. A truncated form of furin (amino acids 26-595) is expressed with a PR-1a signal peptide at N-terminus and His6-KDEL at C-terminus | Homo sapiens |
Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
extracellular | the enzyme contains a signal peptide (amino acids 1-25) and is secreted | Homo sapiens | - |
- |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Ca2+ | required | Homo sapiens |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
factor IX + H2O | Homo sapiens | - |
? | - |
? | |
PA83 + H2O | Homo sapiens | a protective antigen | ? | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Homo sapiens | P09958 | - |
- |
Posttranslational Modification | Comment | Organism |
---|---|---|
glycoprotein | N-glycosylation is necessary for the proper folding and catalytic activity of plant produced or commercial human furin (NEB). Plant-produced human furin treated with PNGase F and Endo H does not exhibit cleavage activity. Similarly, when commercial human furin (NEB) is treated with commercial Endo H or PNGase F, no cleavage is observed for the substrate PA83 | Homo sapiens |
Purification (Comment) | Organism |
---|---|
recombinant His6-tagged enzyme from Nicotiana benthamiana leaves by nickel affinity chromatography, co-purification of the coexpressed proteins | Homo sapiens |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
factor IX + H2O | - |
Homo sapiens | ? | - |
? | |
additional information | to transiently express the substrate factor IX (FIX, amino acids 29-461) in Nicotiana benthamiana plants, the signal peptide (amino acids 1-28) is removed from the FIX sequence (UniProt ID P00740) and replaced with a Nicotiana tabacum PR-1a signal peptide. a KDEL sequence and the His6 tag are added to the C-terminus. Enzyme substrate precursor polypeptide of factor IX (FIX) undergoes several post translational modifications (PTMs), including the removal of the signal peptide (aa 1-28), carboxylation of the first 12 glutamic acid residues downstream from the 18-amino acid propeptide sequence (aa 29-46) in the region rich in glutamic acid (aa 47-92, called the gamma-carboxyglutamic acid or Gla domain) at the N-terminus. Proper gamma-carboxylation of the Gla domain is required for binding to calcium and phospholipids that is critical for proper protease activity during coagulation. In vivo, vitamin K-dependent gamma-carboxylase binds to the 18-amino acid propeptide of FIX, which is then cleaved and is required for optimal binding of the Gla domain to Ca2+ and phospholipids | Homo sapiens | ? | - |
? | |
PA83 + H2O | the protective antigen substrate is from Bacillus anthracis, commercial plant-produced deglycosylated PA83 (dPA83). Cleavage of the substrate generates two protein fragments with distinctly different molecular masses of 63 kDa (PA63) and 20 kDa (PA20). Plant-produced PA83 protein is almost fully cleaved by commercial furin, while plant-produced truncated enzymatically active furin displays about 75% relative activity compared to commercial human furin in vitro | Homo sapiens | PA63 + PA20 | - |
? | |
PA83 + H2O | a protective antigen | Homo sapiens | ? | - |
? | |
protein APRIL + H2O | commercial substrate preparation | Homo sapiens | ? | - |
? |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Homo sapiens |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Homo sapiens |
General Information | Comment | Organism |
---|---|---|
malfunction | plant-produced human furin treated with PNGase F and Endo H does not exhibit cleavage activity. Similarly, when commercial human furin (NEB) is treated with commercial Endo H or PNGase F, no cleavage is observed for the substrate PA83 | Homo sapiens |
physiological function | furin is a ubiquitous proprotein convertase that is involved in the processing (activation) of a wide variety of precursor proteins, including blood coagulation factors, cell surface receptors, hormones and growth factors, viral envelope glycoproteins, etc. and plays a critical regulatory role in a wide variety of cellular events | Homo sapiens |