1. Two General Methods for the Isolation of Enzyme Activities by Colony Filter Screening 
Christian Heinis, Samu Melkko, Salvatore Demartis, Dario Neri 
Chemistry & Biology 
Volume 9, Issue 3, Pages (March 2002)
DOI: /S (02)

2. Figure 1
Schematic Representation of the Filter Sandwich Assay for a Biotin Ligase
(A) Bacterial colonies expressing enzyme on a hydrophilic filter on top of a solid medium.
(B) Bacteria on the master filter release biotin ligase (BirA) to the reaction filter, which is coated with a fusion protein of GST, and the biotin acceptor peptide (BAP), which represents the substrate of the reaction.
(C) Fusion protein of BirA-calmodulin-ABD-His6 is released from the bacteria and immobilized on the human serum albumin (HSA)-coated reaction filter by the noncovalent interaction of the albumin binding domain (ABD) and HSA.
Chemistry & Biology 2002 9, DOI: ( /S (02) )

3. Figure 2
Schematic Representation of the Biotin Ligase (BirA) Activity Assay
(A) BirA biotinylates the immobilized biotin acceptor peptide (BAP) in a buffer supplied with biotin and ATP. The biotinylated peptide representing the product is detected by a streptavidin-HRP (horseradish peroxidase) conjugate.
(B) The fusion protein BirA-CaM-ABD-His6 is immobilized on the reaction filter, which is coated with human serum albumin (HSA). The substrate peptide, linked to a high-affinity calmodulin binding peptidic moiety, is then added to BirA-CaM-ABD-His6 and binds to calmodulin. BirA biotinylates the peptide, and the product is detected with streptavidin-HRP.
Chemistry & Biology 2002 9, DOI: ( /S (02) )

4. Figure 3 Vectors for the Cytoplasmic Expression of BirA
(A) Hexa-histidine-tagged BirA.
(B) Hexa-histidine-tagged BirA-calmodulin-ABD fusion protein.
ABD stands for albumin binding domain; amp stands for ampicillin resistance gene; and BirA stands for biotin ligase gene.
Chemistry & Biology 2002 9, DOI: ( /S (02) )

5. Figure 4
SDS-PAGE Analysis of Proteins Expressed in the Cytoplasm of E. coli
(A) Lane 1: molecular-weight marker. Lane 2: hexa-histidine-tagged BirA (from pCHH20/TG1) purified by nickel affinity chromatography. Lane 3: hexa-histidine-tagged fusion protein of BirA, calmodulin, and albumin binding domain (from pCHH19/TG1) purified by nickel affinity chromatography.
(B) Lane 1: molecular-weight marker. Lane 2: biotin acceptor protein (BAP) fused to the C terminus of GST via an 8 amino acid linker (from pCHH21/TG1) purified by glutathione sepharose affinity chromatography.
Chemistry & Biology 2002 9, DOI: ( /S (02) )

6. Figure 5
catELISA Experiments in which the Two Different Assay Formats from Figure 2 Were Used
(A) lane 1, no enzyme was added to the reaction (negative control 1); lane 2, BirA-His6 was added to the reaction mixture; lane 3, BirA-calmodulin-ABD-His6 fusion protein was added to the reaction mixture; lane 4, BirA-His6 was added to the reaction mixture, devoid of biotin (negative control 2); lane 5, BirA-His6 was added to the reaction mixture, devoid of ATP (negative control 3); lane 6, BirA-His6 was added to the reaction mixture, but the microtiter well was coated with GST (negative control 4).
(B) lane 1, no BirA-calmodulin-ABD-His6 fusion protein was added to the reaction mixture (negative control 1); lane 2, BirA-calmodulin-ABD-His6 fusion protein and biotinylated peptide were added to the reaction mixture (positive control); lane 3, BirA-calmodulin-ABD-His6 fusion protein and substrate peptide were added to the reaction mixture; lane 4, BirA-calmodulin-ABD-His6 fusion protein and substrate peptide were added to the reaction mixture, and soluble BirA-His6 was also added; lane 5, BirA-calmodulin-ABD-His6 fusion protein and substrate peptide were added to the reaction mixture, devoid of biotin (negative control 2); lane 6, BirA-calmodulin-ABD-His6 fusion protein and substrate peptide were added to the reaction mixture, devoid of ATP (negative control 3). See also the Results and Experimental Procedures sections.
Chemistry & Biology 2002 9, DOI: ( /S (02) )

7. Figure 6
Two Different Filter Sandwich Activity Assays for the Biotin Ligase
The figure shows X-ray films, which were exposed to the reaction filters treated with streptavidin-HRP and the chemiluminescence ECL detection kit. The schematic drawing above each film illustrates the reaction conditions.
(A) Left filter, BirA-His6 released from bacterial cells biotinylates the BAP in the presence of biotin and ATP (1 min exposure time); middle filter, the reaction filter is coated with GST that does not have a BAP fused to it (negative control 1; 1 min exposure time [the same filter was still negative for exposures as long as 30 min]); right filter, bacteria do not express biotin ligase (negative control 2; 1 min exposure time).
(B) Left filter, BirA-calmodulin-ABD-His6 is released from the bacterial cells, and biotinylated peptide is added to the reaction (positive control; 30 s exposure time); middle filter, substrate peptide is added to the reaction (exposure time: 20 min); right filter, no peptide is added to the reaction (exposure time: 20 min). For further details, see the Results section.
Chemistry & Biology 2002 9, DOI: ( /S (02) )