Stimulation of chymosin secretion by simultaneous expression with chymosin-binding llama single-domain antibody fragments in yeast.
We studied the effect of coexpression of chymosin and chymosin-binding llama single-domain antibody fragments (VHHs) on the secretion of chymosin by Saccharomyces cerevisiae cells. A VHH expression library containing chymosin-specific VHHs was obtained by immunization of a llama and coexpressed with chymosin in yeast. From this library, we obtained two VHH clones that stimulated chymosin secretion by screening colonies for the level of chymosin secreted. These VHHs bound biotinylated chymosin in an immunoblot procedure but failed to bind chymosin in ELISA, suggesting that their interaction with chymosin was of low affinity. In a second approach, chymosin-specific VHHs were first selected using phage display and then coexpressed with chymosin in yeast cells. Screening yeast cells for higher levels of chymosin secretion resulted in 11 VHHs. Sequence analysis revealed that these 11 VHHs formed four sets of related VHHs that were different from the previously isolated two VHHs. Although binding of VHHs to chymosin could not be demonstrated in ELISA using soluble VHHs, it could be unambiguously demonstrated for clones isolated by phage display, using phage-displayed VHHs. Finally, quantitative Western blot analysis of chymosin amounts demonstrated that coexpression with VHH domains can stimulate the level of secreted chymosin 1.5- to 6-fold.
Harmsen
M M
MM
Institute for Animal Science and Health (ID-Lelystad), Department of Notifiable Diseases and Pathology, Edelhertweg 15, 8219 PH Lelystad, The Netherlands. m.m.harmsen@id.wag-ur.nl
Smits
C B
CB
de Geus
B
B
D000818:Animals; D000915:Antibody Affinity; D000918:Antibody Specificity; D000946:Antigens, Fungal / Q000276:immunology; D001709:Biotechnology / Q000379:methods; D015153:Blotting, Western; D002161:Camelids, New World; D012085:Chymosin / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000276:immunology; D019151:Peptide Library; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA
D000946:Antigens, Fungal; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011994:Recombinant Proteins; D012085:Chymosin