HuProt™ technical details.
CDI Labs’ HuProt™ Human Proteome Microarray is the most comprehensive human full-length protein array ever created (Jeong et al, 2012). Our latest version, HuProt™v4.0, contains over 21,000 human proteins and protein isoforms. This covers over 81% of human proteins in each major functional category of the proteome, as defined by the Human Protein Atlas (Venkataraman et al, 2018), and allows hundreds of interactions to be profiled at once in a wide range of applications, including mapping antigen-specific immunity (multi-isotype serum profiling), determining monoclonal antibody specificity, and to study protein-protein interaction, substrate identification, protein-DNA binding, protein-RNA binding, and binding of some small molecules.
The HuProt™ expression library was created by inserting full-length human open reading frames (ORFs) inserted into a yeast high-copy expression vector that produces GST-His6 fusion proteins when induced in yeast (Hu S et al 2009; Jeong J et al 2012). Each human ORF expression vector was verified for integrity by Sanger sequencing (Venkataraman et al, 2018). These sequence-verified expression vectors encoding human ORFs were transformed into yeast to create our master library; this eukaryotic expression system ensures HuProt™ proteins maintain both function and proper conformation (Hu S, poster).
As required — about once per year — this yeast library is used to synthesize human proteins in high-throughput. Full-length synthesized proteins are purified via their GST-His6 fusion tags as previously described (Hu S et al, 2009). These proteins are then frozen in aliquots in a 384-well format for later use in printing on HuProt™ microarrays.
To print individual batches of HuProt™ arrays, the purified human proteins are then printed as duplicate spot pairs using an Arrayjet UltraMarathon printer (Arrayjet, UK) on PATH nitrocellulose slides (GraceBio, USA) . Controls on HuProt™ include titrated GST protein, titrated human IgG, other human antibody isotypes, histones, mouse and rabbit anti-biotin, mouse IgM, and biotin-tagged control for streptavidin detection. HuProt™ also contains landmark control spots, including Alex Fluor 555/647 (Jeong et al, 2012). Success of each batch of HuProt™ microarrays is validated by anti-GST staining. This confirms full-length GST-His6 fusion proteins were successfully expressed, synthesized, purified, and printed across our clone collection.