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Journal of Biochemistry Advance Access originally published online on November 24, 2007
Journal of Biochemistry 2007 142(6):665-669; doi:10.1093/jb/mvm226
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© 2007 The Japanese Biochemical Society.

Rapid Communication

Production of a Recombinant Fab in Pichia pastoris from a Monocistronic Expression Vector

Rafael Trindade Burtet1,2, Marcos Antônio Santos-Silva1, Guilherme Antônio Marques Buss1, Lidia Maria Pepe Moraes1, Andrea Queiroz Maranhão1,2 and Marcelo Macedo Brigido1,2,*

1Universidade de Brasília, 70910-900, Brasília, DF, Brazil; and 2Institute for Investigation in Immunology, Millennium Institutes, MCT, Brasília, Brazil

*To whom correspondence should be addressed. Tel: 55 (61) 3072423, Fax: 55 (61) 3498411, E-mail: brigido{at}unb.br

Received October 16, 2007; Accepted November 15, 2007


  Abstract

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.

Key Words: antibody engineering, fab expression, kex, Pichia pastoris

Abbreviations: BMGY, buffered glycerol-complex medium; CH1, first heavy chain constant domain; Ck, kappa light chain constant domain; Fab, antibody's antigen binding site; Fd, heavy chain of Fab fragment; Kex, kexin-like; ORF, open reading frame; PMSF, phenylmethanesulphonyl fluoride; rFab, recombinant Fab; scFab, single chain Fab; scFv, single chain variable fragment; L, Light chain of Fab; VL, light chain variable domain


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