RESUMO
A system for the production of mutant recombinant human alpha-fetoprotein (rhAFPO) lacking the glycosylation site has been engineered in the yeast Pichia pastoris. A strain of the methylotrophic yeast Pichia pastoris GS 115/pPICZ?A/rhAFP0, which produces unglycosylated rhAFPO and secretes it to the culture medium, has been constructed. Optimization and scale-up of the fermentation technology have resulted in an increase in the rhAFP0 yield to 20 mg/L. A scheme of isolation and purification of biologically active rhAFP0 has been developed. The synthesized protein has the antitumor activity, which is analogous to the activity of natural human embryonic alpha-fetoprotein.
Assuntos
Proteínas Mutantes/biossíntese , Neoplasias/tratamento farmacológico , Proteínas Recombinantes/biossíntese , alfa-Fetoproteínas/biossíntese , Linhagem Celular Tumoral , Fermentação , Humanos , Proteínas Mutantes/administração & dosagem , Proteínas Mutantes/genética , Pichia/genética , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Saccharomyces cerevisiae/genética , alfa-Fetoproteínas/administração & dosagem , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/isolamento & purificaçãoRESUMO
The yeast strain Pichia pastoris, a producer of humanized F(ab')2 fragments of rabies-blocking antibodies, has been obtained. Human chaperone BiP coexpression caused a twofold increase of the immunoglobulins secretion level. The use of Fos and Jun zippers in the composition of heavy chains facilitated the dimerization of F(ab')2 fragments of the shared pool of secreted immunoglobulins up to 75%.
Assuntos
Anticorpos Monoclonais Humanizados , Anticorpos Antivirais , Fragmentos Fab das Imunoglobulinas , Pichia/metabolismo , Vírus da Raiva , Anticorpos Monoclonais Humanizados/biossíntese , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/isolamento & purificação , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/química , Anticorpos Antivirais/genética , Anticorpos Antivirais/isolamento & purificação , Fragmentos Fab das Imunoglobulinas/biossíntese , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Pichia/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
We have studied the efficiency of N-terminal processing of the antibody light chain depending on the structure of the leader sequence when expressed in the yeast Pichia pastoris. The humanized light kappa-chain of the murine antibody H3-1 and the Saccharomyces cerevisiae alpha-factor pre-pro-leader sequence (pre-pro-alpha-F) were used as models. The use of pre-region of the pre-pro-alpha-F alone or together with the Glu-Ala-linker leads to the slightly increased yield of the secreted L-chain but was accompanied by the incomplete N-terminal processing of the secreted product.
