Soshana Svendsen1, Erin McMillan2, Chad Zimprich1, Elizabeth Capowski2, Mark McDougall3, Dieter Klaubert3, Clive N. Svendsen2, Georgyi V. Los1
1Research and Development, Promega Corporation, Madison, WI 53711
2Waisman Center, University of Wisconsin, Madison, WI 53705
3Promega Biosciences, Inc., San Luis Obispo, CA 93401
We demonstrate that the HaloTag® Protein Reporter can be fused to a truncated human integrin β1 subunit (trβ1IntHT2™) at the amino terminus for extracellular localization. We used a non-permeable green fluorophore to distinguish the cell surface-expressed pool for studying protein internalization and a separate permeable red fluorophore to distinguish the internal pool for studying protein trafficking. With both spatial and temporal separation of protein pools in live cells, we can simultaneously show that the trβ1IntHT2™ fusion protein is properly trafficked to and from the plasma membrane. In addition, labeling the trβ1IntHT2™ protein in live cells followed by fixation and immunocytochemistry for different subcellular markers shows that the fusion protein distributes normally with protein trafficking compartments (e.g., ER and golgi) and protein recycling machinery (e.g., endosomes) and also shows that the fusion protein retains some expected protein co-localizations.
We further show that the HaloTag® Protein and fusion proteins can be expressed in human neural progenitor cells and can be labeled with different fluorophores. The HaloTag® Protein is well tolerated and expressed in the expected pattern by human neural progenitor cells. These results indicate that the HaloTag® Technology is a useful tool for understanding protein trafficking, recycling, and subcellular distribution in an array of mammalian cells, including human neural progenitor cells.