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. 2013 Feb 8;20(5):698–708. doi: 10.1038/cdd.2012.165

Figure 6.

Figure 6

Production of recombinant LBP/p40 in different expression systems and reactivity with mCD14. (a) Presence of recombinant protein in LBP/p40 (p40)-transformed or non-transformed (NT) E. coli lysates was detected by probing immunoblots of total protein with mAb 15308. (b) LBP/p40 was expressed as an intracellular protein in E. coli and MCF-7 cells or as a secreted protein in K562 cells. Immunoblots of nickel affinity-purified LBP/p40 preparations were probed with mAb 15308 (left panel), Lam-R (a commercially available anti-LBP/p40 antibody; middle panel) or anti-V5 (right panel). (c) Expression of recombinant LBP/p40 in baculovirus-infected sf9 insect cells detected by probing immunoblots of total protein from non-infected (NI) or recombinant-LBP/p40 (p40) baculovirus-infected cells with mAb 15308. Nickel metal affinity-purified LBP/p40 (p40/F) is shown for comparison. In all immunoblots, equivalent amounts of total protein were included in each lane. (d) Flow cytometric analysis of binding of purified preparations of recombinant LBP/p40 produced in E. coli to wild-type K562 cells (CD14 negative) or to K562 cells overexpressing CD14 (CD14 positive). Bound recombinant protein was detected with anti-V5 antibody followed by anti-mouse secondary antibody labelled with FITC (GAM-FITC). (e) Failure of contaminating LPS to mediate binding of recombinant LBP/p40 to CD14. Purified preparations of LPS-free, eukaryotic LBP/p40, prepared from MCF-7 or K562 expression systems were incubated with CD14 negative or CD14 positive K562 cells and binding visualised as in (d). Binding of recombinant protein preparations were assessed in the presence or absence of LPS in concentration equivalent to that found contaminating the bacterial preparation used in (d). (f) LBP/p40 derived from a baculovirus insect expression system fails to bind preferentially to mCD14-expressing cells. Purified preparations of insect-derived LBP/p40 were tested for binding to mCD14 negative and positive cells as described for (d) and (e). Numbers at the top right of each histogram represent the total protein concentration of recombinant LBP/p40 incubated with cells (μg/ml). Surface expression of CD14 in K562 (d–f) cells was monitored by staining with mAb 63D3 (anti-CD14)