New Genetic Constructs for Generation of Stable Therapeutic Antibodies to Organophosphorus Toxins in Methylotrophic Yeasts Pichia Pastoris.
We propose a new method of obtaining of stable Fab-fragments of antibodies in Pichia pastoris expression system. Recently, we obtained Fab-fragments of antibodies neutralizing organophosphorus toxins. However, high yield of the target products was not attained because of high level of proteolytic degradation. In the present study, we identified sites of proteolytic degradation in Fab-fragments and endogenous proteases performing degradation, which allowed obtaining optimized genetic constructs for expression of antibody heavy chains (IgGγ1) and kappa and lambda isotypes of light chains. Co-transformation of these vectors allowed obtaining Fab-fragments of antibodies to organophosphorus toxins without proteolytic degradation of the product.
Laboratory of Biocatalysis, M. M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.
Yu A
YA
Mokrushina
Laboratory of Biocatalysis, M. M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.
A V
AV
Stepanova
Laboratory of Biocatalysis, M. M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.
T V
TV
Bobik
Laboratory of Biocatalysis, M. M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia. smirnov@mx.ibch.ru.
I V
IV
Smirnov
Laboratory of Biocatalysis, M. M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.
A G
AG
Gabibov
D000595:Amino Acid Sequence; D017151:Antibodies, Catalytic / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D005656:Fungal Proteins / Q000502:physiology; D015870:Gene Expression; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D009943:Organophosphorus Compounds / Q000037:antagonists & inhibitors*; D010447:Peptide Hydrolases / Q000502:physiology; D010843:Pichia / Q000201:enzymology / Q000235:genetics*; D015202:Protein Engineering; D059748:Proteolysis
D017151:Antibodies, Catalytic; D005656:Fungal Proteins; D007140:Immunoglobulin Fab Fragments; D009943:Organophosphorus Compounds; D010447:Peptide Hydrolases
Fab-fragments of antibodies; catalytic antibodies; expression system in Pichia pectoralis; isolation of recombinant proteins; organophosphorus substances