Fig. 1

Construction and expression of scFvCD16A-sc4-1BBL. a Schematic diagram of the molecular structure scFvCD16A-sc4-1BBL. scFvCD16A-sc4-1BBL was composed of a single chain variable fragment of human CD16A antibody, three extracellular domains (ECDs) of 4-1BBL and a 6X His tag at the C-terminus. b The coding sequence of scFvCD16A-sc4-1BBL was inserted into the pGAPzα-rDNA plasmid under the control of the GAP promoter, which then integrated into the genome of K. phaffii X33 through homologous recombination. c The positive colones expressing scFvCD16A-sc4-1BBL were screened by dot blotting using an anti-His tag HRP conjugate antibody. d The high expression clones were further identified by Western blotting under nonreducing conditions (-DTT) using the same antibody as c. The clone of No. # 72–3 showed the highest expression and was selected for the following study