Identification, gene cloning and expression of serine proteases in the extracellular medium of Nicotiana tabacum cells.
Recombinant proteins secreted from plant suspension cells into the medium are susceptible to degradation by host proteases secreted during growth. Some degradation phenomena are inhibited in the presence of various protease inhibitors, such as EDTA or AEBSF/PMSF, suggesting the presence of different classes of proteases in the medium. Here, we report the results of a proteomic analysis of the extracellular medium of a Nicotiana tabacum bright yellow 2 culture. Several serine proteases belonging to a Solanaceae-specific subtilase subfamily were identified and the genes for four cloned. Their expression at the RNA level during culture growth varied depending on the gene. An in-gel protease assay (zymography) demonstrated serine protease activity in the extracellular medium from cultures. This was confirmed by testing the degradation of an antibody added to the culture medium. This particular subtilase subfamily, therefore, represents an interesting target for gene silencing to improve recombinant protein production.                         Key message The extracellular medium of Nicotiana tabacum suspension cells contains serine proteases that degrade antibodies.
Institut des Sciences de la Vie, Université catholique de Louvain, Croix du Sud 4-5, 1348, Louvain la Neuve, Belgium.
Catherine
C
Navarre
Benoit
B
De Muynck
Georges
G
Alves
Didier
D
Vertommen
Bertrand
B
Magy
Marc
M
Boutry
D000818:Animals; D002417:Cattle; D018929:Cell Culture Techniques; D003001:Cloning, Molecular / Q000379:methods*; D003470:Culture Media / Q000378:metabolism*; D004789:Enzyme Activation; D015870:Gene Expression; D017343:Genes, Plant; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D010802:Phylogeny; D059828:Plant Cells / Q000201:enzymology*; D059748:Proteolysis; D020543:Proteome / Q000032:analysis; D018749:RNA, Plant / Q000235:genetics; D057057:Serine Proteases / Q000235:genetics / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics
D003470:Culture Media; D007074:Immunoglobulin G; D020543:Proteome; D018749:RNA, Plant; D012710:Serum Albumin, Bovine; D057057:Serine Proteases; D013381:Subtilisins; C119297:subtilase, plant