Inhibition of protease activity by antisense RNA improves recombinant protein production in Nicotiana tabacum cv. Bright Yellow 2 (BY-2) suspension cells.
Recombinant proteins produced in plant suspension cultures are often degraded by endogenous plant proteases when secreted into the medium, resulting in low yields. To generate protease-deficient tobacco BY-2 cell lines and to retrieve the sequence information, we cloned four different protease cDNAs from tobacco BY-2 cells (NtAP, NtCP, NtMMP1, and NtSP), which represent the major catalytic classes. The simultaneous expression of antisense RNAs against these endogenous proteases led to the establishment of cell lines with reduced levels of endogenous protease expression and activity at late stages of the cultivation cycle. One of the cell lines showing reduced proteolytic activity in the culture medium was selected for the expression of the recombinant full-length IgG1(κ) antibody 2F5, recognizing the gp41 surface protein of HIV-1. This cell line showed significantly reduced degradation of the 2F5 heavy chain, resulting in four-fold higher accumulation of the intact antibody heavy chain when compared to transformed wild type cells expressing the same antibody. N-terminal sequencing data revealed that the antibody has two cleavage sites within the CDR-H3 and one site at the end of the H4-framework region. These cleavage sites are found to be vulnerable to serine proteases. The data provide a basis for further improvement of plant cells for the production of recombinant proteins in plant cell suspension cultures.
RWTH Aachen University, Institute for Molecular Biotechnology, Aachen, Germany.
Manoj K
MK
Mandal
Rainer
R
Fischer
Stefan
S
Schillberg
Andreas
A
Schiermeyer
D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000080908:Broadly Neutralizing Antibodies; D018929:Cell Culture Techniques; D015483:HIV Antibodies; D015700:HIV Envelope Protein gp41 / Q000378:metabolism; D007145:Immunoglobulin kappa-Chains / Q000378:metabolism; D008958:Models, Molecular; D010447:Peptide Hydrolases / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000378:metabolism; D017433:Protein Structure, Secondary; D016372:RNA, Antisense / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics
C558560:2F5 monoclonal antibody; D000911:Antibodies, Monoclonal; D000080908:Broadly Neutralizing Antibodies; D015483:HIV Antibodies; D015700:HIV Envelope Protein gp41; D007145:Immunoglobulin kappa-Chains; D010940:Plant Proteins; D016372:RNA, Antisense; D011994:Recombinant Proteins; D010447:Peptide Hydrolases
Antisense RNA; BY-2 cells; Gene silencing; Plant molecular farming; Recombinant protein degradation