Metabolic Biotinylation of Recombinant Proteins in Mammalian Cells and in Mice  M.Brandon Parrott, Michael A. Barry  Molecular Therapy  Volume 1, Issue 1, Pages 96-104 (January 2000) DOI: 10.1006/mthe.1999.0011 Copyright © 2000 American Society for Gene Therapy Terms and Conditions

Fig. 1 Alignment of PSTCD and deletion constructs to other mammalian biotinylated proteins. ClustalW alignment was performed on the PSTCD, mouse pyruvate carboxylase, and human acetyl-CoA carboxylase. The biotinylated lysine in each protein is indicated by ˙. Shaded boxes indicate identical amino acids between proteins. N-terminal and C-terminal deletions are indicated by pointing in the direction of the sequence remaining in the deletion construct. Amino acid numbers (70AA, 63AA, 56AA, 43AA, and 93AA) designate the size of PSTCD fragments remaining in the deletion constructs described in the text. Molecular Therapy 2000 1, 96-104DOI: (10.1006/mthe.1999.0011) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

Fig. 2 Mammalian cell biotinylation of a prokaryotic carboxylase domain fused to EGFP and Ad5 Knob. Mammalian expression vectors were constructed in order to express a bacterially biotinylated carboxylase domain (PSTCD) as a fusion to the C-terminus of EGFP and an EGFP–KNOB adenovirus type 5 fusion protein. The resulting recombinant proteins were predicted from their sequences to have molecular weights of 42.8 and 64.3 kDa, respectively. (A) Cartoon of expressed fusion proteins. (B) Western blot analysis of total cell lysates from transfected CHO cells. Cells were lysed 24 h following transient transfection, run on 7.5% Tricine–SDS–PAGE gels, and blotted on PVDF membranes. EGFP fusions and biotinylated proteins were detected with monoclonal anti-GFP and avidin–HRP. Biotin (1 and 100 μμ) indicates the level of biotin in the culture after transfection. 1 μμ is the normal biotin level in RPMI 1640 tissue culture media. Molecular Therapy 2000 1, 96-104DOI: (10.1006/mthe.1999.0011) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

Fig. 3 Minimization of PSTCD in mammalian cells in different protein contexts. Sequential truncations of the PSTCD linked to the C-terminus of EGFP and the KNOB protein were made by long PCR mutagenesis. These constructs were then transfected into CHO cells and analyzed by Western blot to determine the minimal PSTCD domain required for biotinylation in mammalian cells. (A) Cartoon of EGFP-PSTCD truncations. (B) Western blots of cells transfected with EGFP-PSTCD truncation constructs as described in the legend to Fig. 2. (C) Cartoon of EGFP-KNOB-PSTCD truncations. The MYC box indicates constructs in which the 11-amino-acid c-myc tag was inserted between KNOB and PSTCD. (D) Western blots of cells transfected with EGFP-KNOB-PSTCD truncation constructs. 10 and 60 sec exposure refer to the length of chemiluminescent detection for the indicated blots. Longer exposure reveals lower amounts of protein. Molecular Therapy 2000 1, 96-104DOI: (10.1006/mthe.1999.0011) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

Fig. 4 Nondenaturing purification of biotinylated recombinant fusion proteins from mammalian cells. CHO cells were transfected with EGFP and EGFP-PSTCD mammalian expression vectors. The cells were harvested 24 h following transfection, lysed by sonication, and purified with the use of monomeric avidin. Samples from the purification protocol were loaded and electrophoresed on 7.5% Tricine-SDS-PAGE gels and analyzed by Coomassie blue staining and Western blot after SDS-PAGE. Coomassie indicates staining of total protein from samples during protein purification. Western blot analysis with avidin-HRP reveals proteins containing biotin. Western blot analysis with anti-GFP antibody reveals proteins containing GFP. Total lysate refers to the whole-cell lysate produced by sonication that was introduced onto the monomeric avidin resin. Unbound refers to those proteins from the whole-cell lysate that were not bound by the avidin resin. Wash 1, 2, and 3 refer to the fractions of sequential washes in which nonspecific proteins were removed from the avidin resin by incubation in cell lysis buffer. Biotin elution refers to the fraction of proteins released from the monomeric avidin resin by incubation with 5 mM biotin. Molecular Therapy 2000 1, 96-104DOI: (10.1006/mthe.1999.0011) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

Fig. 5 Production of recombinant biotin-labeled proteins in mice. Female BALB/c mice were transfected with plasmid DNA by naked DNA injection into the skin. (A) GFP fluorescence in injection sites 24 h after plasmid introduction. The skin was visualized in living mice at 20× magnification at the injection sites in the tails of the animals. The punctate GFP fluorescence observed is characteristic of individual transfected cells viewed under low magnification (1). (B) Western blot of tail tissue sections from the injection sites shown in A using avidin–HRP to detect biotinylated proteins in the mouse tissue. Molecular Therapy 2000 1, 96-104DOI: (10.1006/mthe.1999.0011) Copyright © 2000 American Society for Gene Therapy Terms and Conditions