The monoclonal antibody market represents the fastest-growing segment within the biopharmaceutical industry. Indeed, recombinant antibodies and antibody fragments are widespread tools for research, diagnostics and therapy. Large-scale production of recombinant antibodies and antibody fragments requires a suitable expression system which has to be cheap, accessible for genetic modifications, easily scaled up. Although prokaryotic expression systems can reduce production costs, recombinant antibody production in conventional bacterial hosts, such as Escherichia coli, often results in inclusion bodies formation. Since lowering of the expression temperature can increase product solubility facilitating its correct folding, a novel process for recombinant antibody fragments production at low temperatures was established by using the Antarctic Gram-negative bacterium Pseudoalteromonas haloplanktis TAC125 as recombinant expression host. To test the versatility of the newly developed process, the production of three aggregation prone model proteins, corresponding to the most common formats of antibody fragments: Fab, scFv and VHH, was evaluated. The construction of an ad hoc genetic expression system for each model protein followed a rational design where several critical aspects were considered including the selection of molecular signals for targeting the proteins to the periplasmic space and the choice of optimal gene-expression strategies. For Fab fragment production in heterodimeric form an artificial operon was designed and constructed. Moreover, a new defined minimal medium was made-up to maximize bacterial growth parameters and recombinant production yields. All model proteins were obtained in soluble and biologically competent form. The observed proficiency of the Antarctic bacterium to produce recombinant antibody fragments was related to the unusually high number of genes encoding peptidyl prolyl cis-trans isomerases found in the P. haloplanktis TAC125 genome, making this bacterium the host of choice for the recombinant production of this protein class.

La produzione di anticorpi ricombinanti nel batterio antartico Pseudoalteromonas haloplanktis TAC125 / Maria, Giuliani; Parrilli, Ermenegilda; Filomena, Sannino; Gennaro Antonio, Apuzzo; Tutino, MARIA LUISA; Gennaro, Marino. - 37:(2014), pp. 87-112. [10.4399/97888548717175]

La produzione di anticorpi ricombinanti nel batterio antartico Pseudoalteromonas haloplanktis TAC125

PARRILLI, ERMENEGILDA;TUTINO, MARIA LUISA;
2014

Abstract

The monoclonal antibody market represents the fastest-growing segment within the biopharmaceutical industry. Indeed, recombinant antibodies and antibody fragments are widespread tools for research, diagnostics and therapy. Large-scale production of recombinant antibodies and antibody fragments requires a suitable expression system which has to be cheap, accessible for genetic modifications, easily scaled up. Although prokaryotic expression systems can reduce production costs, recombinant antibody production in conventional bacterial hosts, such as Escherichia coli, often results in inclusion bodies formation. Since lowering of the expression temperature can increase product solubility facilitating its correct folding, a novel process for recombinant antibody fragments production at low temperatures was established by using the Antarctic Gram-negative bacterium Pseudoalteromonas haloplanktis TAC125 as recombinant expression host. To test the versatility of the newly developed process, the production of three aggregation prone model proteins, corresponding to the most common formats of antibody fragments: Fab, scFv and VHH, was evaluated. The construction of an ad hoc genetic expression system for each model protein followed a rational design where several critical aspects were considered including the selection of molecular signals for targeting the proteins to the periplasmic space and the choice of optimal gene-expression strategies. For Fab fragment production in heterodimeric form an artificial operon was designed and constructed. Moreover, a new defined minimal medium was made-up to maximize bacterial growth parameters and recombinant production yields. All model proteins were obtained in soluble and biologically competent form. The observed proficiency of the Antarctic bacterium to produce recombinant antibody fragments was related to the unusually high number of genes encoding peptidyl prolyl cis-trans isomerases found in the P. haloplanktis TAC125 genome, making this bacterium the host of choice for the recombinant production of this protein class.
2014
La produzione di anticorpi ricombinanti nel batterio antartico Pseudoalteromonas haloplanktis TAC125 / Maria, Giuliani; Parrilli, Ermenegilda; Filomena, Sannino; Gennaro Antonio, Apuzzo; Tutino, MARIA LUISA; Gennaro, Marino. - 37:(2014), pp. 87-112. [10.4399/97888548717175]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/587279
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