Fluorescent Citrine-IgG fusion proteins produced in mammalian cells

Abstract

Genetically encoded fluorescent antibodies are desirable for many applications in biotechnology, proteomics, microscopy, cell biology and molecular diagnostics, although efficient production of fluorescent IgGs in mammalian cells has been hampered by different and mutually incompatible secretion- and folding-requirements of antibodies and green fluorescent protein-derived fluorescent entities. Here, we show that this hurdle can be overcome by generating whole antibody fusions with Citrine, a modified yellow fluorescent protein that folds properly in the endoplasmic reticulum of mammalian cells. Applying optimized connector sequences, one or more Citrine molecules can be fused to different positions of IgGs without interfering with folding, secretion or function of the fusion proteins. These proteins can be transiently expressed and purified to similar yields as unmodified antibodies using standard technologies. IgG-Citrine fusions fully retain binding specificity and affinity, and can be applied to assays that require labeled IgG. A particularly interesting feature is the pH-dependency of Citrine fluorescence. This makes IgG-Citrine fusion proteins a valuable tool to track antibody target binding, internalization and subsequent intracellular trafficking to acidic compartments.

Figures and Tables

Figure 1 Composition of antibody-Citrine fusion protein formats. Three general formats were generated: either Citrine was added to the C-termini of the light chains (LC) (top row) or to the C-termini of the heavy chains (HC) (middle row) or Citrine was inserted between two single chain Fvs (scFvs) (bottom row). The two different heavy chain formats both use the “knobs-into-holes” technology. One format uses protein complementation (PCA) to generate a full Citrine molecule from two halves, the other format has full length (FL) Citrine at the C-terminus of only one HC. The format with two scFvs has a Hexa-Histidine tag (6x-His) for purification. The variable antibody regions used targeted either the DIG hapten (anti-DIG) or the IGF-1 receptor (anti-IGF-1R).

Figure 2 Expression and purification of antibody-Citrine fusion proteins. An example for Protein A purification (A) of a DIG-specific antibody with Citrine attached to the C-termini of the light chains from culture supernatants. Grey line pH, brown line conductivity, blue line 280 nm, red line 527 nm. The same protein was subjected to size exclusion chromatography (SEC) purification (B) using a Superdex200 column. Fractions collected between both red lines were pooled and used for further analysis. Blue line 280 nm, brown line conductivity, grey line pH. Analysis of the purified product (blue line) by analytical SEC (C) using a Superdex200 column at 280 nm shows the purity (98.7%) of the antibody-Citrine fusion protein. Reference mix (black line) with (from left) 670 kDa, 158 kDa, 44 kDa, 17 kDa and 1.3 kDa.

Figure 3 Antibody-Citrine fusion proteins retain the full binding specificity and affinity of their parent antibodies. Surface plasmon resonance (SPR) analysis was performed with the antibody formats indicated. All proteins were captured by a anti-human IgG antibody to the chip. To determine the binding affinities of the different formats, mono-digoxygenated myoglobin was injected in increasing concentrations. K_D values were obtained using non-linear curve fitting (Langmuir). Affinities for each format are indicated in nM. Further information can be found in Table 2. Depictions of the molecules are described in Figure 1.

Figure 4 All antibody-Citrine fusion protein variants show pH-dependent Citrine fluorescence. 50 nM of the different antibody-Citrine fusion protein formats were incubated in buffer adjusted to the pH indicated. The sample was excited at 516 nm and the fluorescence was determined at 529 nm. Fluorescence was measured and normalized for all fusion proteins to generate a signal independent of the number of Citrine molecules. An anti-DIG IgG without Citrine was used as a negative control. Depictions of the molecules are described in Figure 1.

Figure 5 Antibody-Citrine fusion proteins to detect cell binding and internalization. Cells were incubated on ice in the presence of an anti-IGF-1R antibody-Citrine fusion protein for 2 h. The cells were either fixed immediately after this period (upper part) or after overnight incubation at 37°C (lower part). A Cy3-labeled human kappa light chain (κLC) specific antibody was used to detect the antibody-Citrine fusion protein. Citrine fluorescence was measured in the FITC channel. Some sub-cellular structures show predominantly Citrine signal (Arrow).

Figure 6 Antibody-Citrine fusion proteins to detect binding to live cells. Cells were incubated in the presence of antibody-Citrine fusion proteins and analyzed by FACS. The left hand parts (A–C) show detection of bound antibodies by APC-labeled secondary antibodies (Sec. AB), the left hand parts show detection by means of the Citrine fluorescence (A–C). The antibody fusion proteins analyzed were (A) full-size anti-IGF-1R antibodies (IGF-1R WT Citrine) and the (B) LeY carbohydrate (LeY WT Citrine), both with Citrine attached to the C-termini of their light chains. A small antibody format (scFv Citrine) with Citrine in line between a scFv specific for the IGF-1 receptor and the DIG hapten was used (C) as an example for a monovalent binding protein. Untreated cells (Cells) were used as the control in all experiments.

Figure 7 Antibody-Citrine fusions to track acidification in living cells. Cells incubated with or without NH₄Cl (NH4) were treated on with an IGF-1 receptor specific antibody carrying Citrine on the C-termini of the light chains (IGF-1R Citrine) for 1 hour on ice. One fraction of both cell populations was analyzed in the FITC channel immediately (A) to monitor Citrine fluorescence. The other fraction was analyzed after 2 h at 37°C (B) to ensure antibody internalization. Untreated cells (Cells) were used as control.

Table 1 Overview of produced recombinant antibodies

Table 2 Overview of binding abilities of DIG-specific Citrine formats