Production of a recombinant Fab in Pichia pastoris from a Monocistronic expression vector.
Recombinant Fab is usually expressed using dicistronic vectors producing the heavy and light chains separately. We developed an improved vector for Fab fragment expression in Pichia pastoris, which allows a stoichiometric expression of both chains based on a monocistronic arrangement. The protein is produced as a unique polypeptide harbouring a KEX2 processing site between both chains. After KEX cleavage, a correctly folded mature Fab is formed. The produced recombinant protein is characterized as a heterodimeric functional Fab. The vector described is a new tool for the proper expression of antibody fragments or any heterodimeric polypeptides.
Burtet
Rafael Trindade
RT
Universidade de Brasília, 70910-900, Brasília, DF, Brazil.
Santos-Silva
Marcos Antônio
MA
Buss
Guilherme Antônio Marques
GA
Moraes
Lidia Maria Pepe
LM
Maranhão
Andrea Queiroz
AQ
Brigido
Marcelo Macedo
MM
D019281:Dimerization; D015870:Gene Expression; D005822:Genetic Vectors; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D007700:Kinetics; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology
D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins