A novel fragment of antigen binding (Fab) surface display platform using glycoengineered Pichia pastoris.
A fragment of antigen binding (Fab) surface display system was developed using a glycoengineered Pichia pastoris host strain genetically modified to secrete glycoproteins with mammalian mannose-type Man(5)GlcNAc(2) N-linked glycans. The surface display method described here takes advantage of a pair of coiled-coil peptides as the linker while using the Saccharomyces cerevisiae Sed1p GPI-anchored cell surface protein as an anchoring domain. Several Fabs were successfully displayed on the cell surface using this system and the expression level of the displayed Fabs was correlated to that of secreted Fabs from the same glycoengineered host in the absence of the cell wall anchor. Strains displaying different model Fabs were mixed and, through cell sorting, the strain displaying more expressed Fab molecule or the strain displaying the Fab with higher affinity for an antigen was effectively enriched by FACS. This novel yeast surface display system provides a general platform for the display of Fab libraries for affinity and/or expression maturation using glycoengineered Pichia.
GlycoFi Inc., A wholly-owned subsidiary of Merck & Co Inc., Lebanon, NH 03766, United States.
Song
S
Lin
Nga Rewa
NR
Houston-Cummings
Bianka
B
Prinz
Renée
R
Moore
Beata
B
Bobrowicz
Robert C
RC
Davidson
Stefan
S
Wildt
Terrance A
TA
Stadheim
Dongxing
D
Zha
D000954:Antigens, Surface / Q000235:genetics / Q000378:metabolism; D002473:Cell Wall / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
D000954:Antigens, Surface; D006023:Glycoproteins; D007140:Immunoglobulin Fab Fragments; D008562:Membrane Glycoproteins; D010455:Peptides; C077394:SED1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins