Metabolic biotinylation of recombinant antibody by biotin ligase retained in the endoplasmic reticulum

Abstract

Due to its strength and specificity, the interaction between avidin and biotin has been used in a variety of scientific and medical applications ranging from immunohistochemistry to drug targeting. The present study describes two methods for biotinylation of proteins secreted from eukaryotic cells using the Escherichia coli biotin protein ligase. In one system the biotin ligase was co-secreted from the cells along with substrate protein enabling extracellular biotinylation of the tagged protein. In the other system, biotin ligase was engineered to be retained in the endoplasmic reticulum (ER) and metabolically biotinylates the secretory protein as it passes through the ER. An engineered antibody fragment, a diabody with specificity for carcinoembryonic antigen (CEA) was fused to the biotin acceptor domain (123 amino acid) of Propionibacterium shermanii. Coexpression of the fusion protein with ER retained biotin ligase showed higher biotinylation efficiency than biotinylation by co-secreted ligase. Biotinylation of the anti-CEA diabody tagged with a short (15 amino acid, Biotin Avitag) biotin acceptor peptide was also successful. Utilization of ER retained biotin ligase for biotinylation of protein is an attractive alternative for efficiently producing uniformly biotinylated recombinant proteins for a variety of avidin-biotin technologies.

Fig. 1

Schematic presentation of the fusion proteins. (A) Anti-CEA diabody fusion proteins. T84.66 V_L and V_H are joined by an 8 aa linker, to form the diabody. 1-4, Diabody variants with 123 aa biotin acceptor domain (BD123) or 15 aa peptide (BP15) at the C-terminus. A 4aa (GSTS) or a 6 aa (GSTSGS) linker were used between Db and biotin acceptor substrate. (B) Biotin protein ligase (BirA). The amino acids DYKD and DYKDEL at the C-terminus are for secreted and ER-retained biotin ligase respectively.

Fig. 2

Western blots of Db-BD123 fusion protein cosecreted with biotin ligase (BirA-DYKD). (A) Expression of secreted BirA-DYKD was detected with anti-FLAG M2 antibody. (B) Expression of Db-BD123 in different clones was detected with HRP-conjugated anti-mouse Fab. (C) Same Db-BD123 expressing clones detected with SA HRP. The molecular weight standard is indicated. C, control, non transfected NS0 cells.

Fig. 3

In vivo biotinylation of Db-BD123 fusion protein cotransfecting with ER-retained biotin ligase (BirA-DYKDEL). (A) Western blot of BirA-DYKDEL expression in total cell lysate (5μg per lane). Expression was detected with anti-FLAG M2 antibody. C, total cell lysate from non transfected NS0 cells. (B) Western blots of media supernatants from NS0 cells expressing Db-BD123 using HRP-conjugated anti-mouse Fab and with SA HRP. C, the supernatant from non transfected NS0 cells. Db, diabody (positive control). (C) Biotinylation efficiency of Db-BD123 by secretory (5D8) and ER-retained (1G7) ligase. Equal amount of proteins were loaded in each lane. Diabody was detected with anti-mouse Fab antibody and biotin was detected with SA HRP.

Fig. 4

Western blots of media supernatants from NS0 cells expressing Db-BP15 using HRP-conjugated anti-mouse Fab and with SA HRP. The presence of 6xHis tag was detected with AP-conjugated anti-His antibody. C, the supernatant from non transfected NS0 cells.

Fig. 5

Biochemical characterization of purified biotinylated anti-CEA diabody fusion proteins. (A) SDS-PAGE. Lane 1, Db-BP15-His: lane 2, Db-BD123; lane 3, Molecular weight markers. (B) Size-exclusion analysis using Superdex 75 HR column. The major peaks eluted at retention times of 18.33 minutes for Db-BD123 and 20.70 minutes for Db-BP15 consistent with dimers of 84 and 60 kDa respectively. The parental anti-CEA diabody [55 kDa; (Wu et al., 1999)] was used as size standard. (C) Size-exclusion analysis of purified Db-BD123 using a Superdex 200 HR column. The major peak eluted at retention time of 28.79 minutes. Intact chimeric T84.66 IgG [150 kDa; (Neumaier et al., 1990)], minibody [80 kDa; (Hu et al., 1996)] and diabody (55 kDa) with the corresponding retention times of 25.8, 28.5 and 38.2 minutes were used as size standards. Proteins were detected by absorbance at 280 nm.

Fig. 6

Efficiency of biotinylation. Western blot using SA HRP to detect binding of biotinylated fusion proteins to streptavidin agarose. Lanes 1 and 4, starting material; lanes 2 and 5, unbound material and lanes 3 and 6, streptavidin bound material.

Fig. 7

Flow cytometry of purified fusion proteins binding to live LS174T-CEA⁺ cells. Cells were incubated with no protein (negative control), in vitro biotinylated chimeric T84.66 antibody (positive control) and purified biotinylated fusion proteins (Db-BD123 and Db-BP15-His). The cells were stained with (A) Alexa Fluor 488 conjugated streptavidin (FL1, horizontal shift) and (B) Qdot655SA (FL3, vertical shift).

Fig. 8

Fluorescence micrographs of LS174T-CEA⁺ cells following incubation with purified fusion protein (Db-BD123 or Db-BP15-His) and Qdot655SA. Cells were imaged in a confocal microscope. LS174T-CEA⁺ cells treated with (A) no protein (negative control), (B) Db-BD123 and (C) Db-BP15-His. The scale bar corresponds to 20 μm.

Fig. 9

Western blots of media supernatants expressing Db-BP15-His in absence of BirA. (A) Expression of diabody was detected with anti-Fab antibody. Lane 1, non transfected NS0 cells (negative control); lanes 2 and 3, cell lines expressing Db-BP15-His in absence of BirA; lane 4, cell line expressing Db-BP15-His in presence of BirA (positive control). (B) Detection of 6xHis tag using AP-conjugated anti-His antibody. Lane 5, non transfected NS0 cells (negative control); lanes 6 and 7, cell lines expressing Db-BP15-His in absence of BirA. (C) Detection of biotin using SA HRP. Lanes 8 and 9, cell lines expressing Db-BP15-His in absence of BirA; lane 7, cell line expressing Db-BP15-His in presence of BirA (positive control).