A Highly Versatile Microscope Imaging Technology Platform for the Multiplex Real-Time Detection of Biomolecules and Autoimmune Antibodies - Molecular Diagnostics - page 55

Fig. 13 Principle of the expression of in-vivo biotinylated GST in E. coli (AVB101): a GST-
encoding cDNA was cloned into the expression vector pan5. Subsequent AVB101 cells were
transformed to overexpress GST including AviTag TM and overexpress biotin ligase which
specifically biotinylates a lysine residue. b Detection principle of biotinylated GST: AVB101
cells expressing either non-biotinylated GST or biotinylated GST (AviTag TM -GST) were grown
in 50 mL LB medium at 37 C. At an OD of 0.7, protein expression was induced (1.5 mM IPTG)
in the presence of 50 lM biotin. After 3 h cells were washed three times with 50 mL ice-cold
water, pelleted and resuspended in 6 mL PBS (50 mM Na-phosphate, pH 7.4, 150 mM NaCl)
including inhibitor cocktail plus (Roth) according to the manufacturer's instructions. Cells were
lysed by French pressing. All cell debris was removed by centrifugation (20 min, 16,000g). The
cleared lysates (200 lL) were incubated on ice for 15 min with about 200 streptavidin-coated
microbeads, which were prepared as described in Rödiger et al. [ 3 ]. The microbeads were then
washed three times with PBST (PBS, 0.1% Tween 20, pH 7.4) by resuspension of microbeads in
200 lL PBST, centrifugation (3 min, 2,250g) and removal of the supernatant. Microbeads were
shaken for 1 h with 25 lL (1:100 in PBS) monoclonal rabbit-anti-GST-antibody (Sigma-Aldrich),
then washed three times with PBST (see above). The microbeads were then incubated with 25 lL
(1:100 in PBST) anti-rabbit-Cy5-labeled secondary antibody (Dianova) for 1h. Finally,
microbeads were washed three times with PBST. c Western blot analysis of expressed GST:
AVB101 was transformed with pan five containing cDNA coding for GST. Samples of 1 mL
culture medium were taken before IPTG induction and after finishing the protein expression.
Lysates of bacteria were analyzed by SDS-PAGE (M, marker in kDa). d VideoScan detection of
biotinylated GST: Bound GST was quantified by Cy5 fluorescence using VideoScan

The VideoScan detection of biotinylated protein/peptide presented here is the
first step in the development of a multiplex protein microbead assay. Building
on this proof-of-principle, we are developing a medium-throughput system for
VideoScan epitope mapping using biotinylated GST-peptide fusion antigens

expressed in E. coli via the AviTag TM -System.