Background

There is hardly a protein to be found within the human body that does not share extensive sequence homology to a family member or isoform, as the human cell requires redundancies to ensure continuous work. Protein isoforms are sometimes encoded by different genes and, often, specific genes have multiple transcripts, returning slightly different proteins depending on how they are spliced. Many diseases are characterized by the predominant expression of specific enzyme isoforms, and isoform expression is often quantified at RNA levels by RT-qPCR, sequencing, or microarrays. Quantifying levels of highly homologous proteins, on the other hand, is difficult and limited by the availability of isoform-specific antibodies. Additionally, batch-to-batch variations in antibody performance as well as recognition of unspecific binding partners sometimes hamper precise protein identification through this method (Voskuil, 2017). Furthermore, when studying newly identified isoforms or lesser studied protein families, there are often no monoclonal, isoform-specific antibodies available. We encountered this issue in our investigation of hexokinase 2 (HK2) and HK3, glycolytic enzymes of the hexokinase family. While sequence homology between HK2 and HK3 is only roughly 54%, we struggled to find appropriate antibodies detecting specifically one isoform or the other when 293T cells were transfected with either HK1, HK2, or HK3 and subsequently stained with a monoclonal antibody for HK2 as well as three antibodies for HK3 (two polyclonal, one monoclonal). The use of polyclonal antibodies should be assessed carefully, as larger immunogens increase the likelihood of producing signals from various family members. If the proteins do not vary in size, their distinction is impossible. HK2 and HK3 are both roughly 100 kDa in size; using antibodies against HK3, we detected signals on western blot in samples that were confirmed HK3-negative through mass spectrometry. This prompted us to generate a protocol for isoform-specific detection of endogenous protein expression without cross-reaction bias.

We hereby suggest the use of an endogenous tag that can be inserted into precise genomic locations and allows relative quantification and identification of protein isoforms through various methods. The system was developed by Promega (Fitchburg, WI, USA) (Schwinn et al., 2018 and 2020). The HiBiT^® peptide tag used is 11 amino acids small and binds tightly to an adaptor protein (LgBiT^®) when supplied. Through this interaction, the bright luminescent protein NanoBiT^® is reconstituted (Dixon et al., 2016). Luminescent intensity from reconstituted NanoBiT is directly proportional to the amount of HiBiT present. This allows for relative protein quantification using a common plate reader. Cell lysate can also be analyzed via SDS gel and subsequent incubation of the membrane with LgBiT^®. Furthermore, Promega recently launched an anti-HiBiT monoclonal antibody, which allows visualizing protein localization and expression within a cell using fluorescent microscopy. The anti-HiBiT antibody can also be used in traditional western blotting.

In order to insert the tag into the genomic locus of interest, CRISPR/Cas9-mediated genome editing is used. For successful editing, a sgRNA that binds very close to the desired insertion of the HiBiT tag is complexed with Cas9, and single-strand donor DNA templates (ssODN) for homology-directed repair are supplemented. A few days after electroporation of the cells with Cas9/sgRNA ribonucleoprotein complex (RNP) and ssODN, cells can be assessed for insertion of the tag using a plate reader. While luminescent signal for bulk edited cells in culture was stable during cell propagation, we recommend growing monoclonal populations with 100% of cells expressing the tag for reliable protein quantification.

With the recently launched anti-HiBiT monoclonal antibody, further downstream experiments such as fluorescent microscopy are possible but will require prior optimization.

This example follows the tagging of two hexokinase proteins (HK2 and HK3), two highly homologous proteins encoded on different chromosomes (Figure 1). Here, we show the results of adding the HiBiT tag to the C-terminal end, but inserting the tag at the N-terminus resulted in the generation of a luminescent signal as well. While this strategy is therefore applicable to splice isoforms that differ in either the first or last exon, we do not currently know whether insertion of the tag within the gene body will allow for binding of the adaptor protein and whether this technique can be applied for splice isoforms that share the first and last exon. Insertion of the tag within the gene body could potentially alter protein folding and/or function, presenting a potential limitation for this technique.

Figure 1. Schematic illustration of tagging options for homologous proteins hexokinase 2 (HK2) and HK3. HiBiT tag can be inserted at either the N- or C-terminal end. Data shown in this protocol are generated with HK2 and HK3 tagged at their C-terminal end.