Producing Functional Proteins

One major challenge for all proteomic arrays is ensuring proper protein functionality. Protein function is directly related to proper folding and post-translational modifications (PTMs), such as phosphorylation. SPOC addresses these challenges to validate the proteins on our arrays in multiple ways.

Validating expression and capture

Each plasmid printed into the DNA nanowells is first sequence-verified to ensure accuracy and identity of every protein on the SPOC array.
We utilize human cell lysate instead of lysate from another organism. This helps ensure that native cellular components are present to assist with expression (such as tRNA abundance, co-factors) and addition of native PTMs (for folding and function). In fact, post-expression glycosylation has been reported for human cell free lysate-expressed proteins (Karthikeyan et al, 2016).
We express proteins with capture tag on either N- or C- terminus, as desired. When capture tag is expressed last and can be detected in the assays, it indicates proper in-frame expression and folding of both protein and the capture tag. Therefore, each SPOC array protein is first assessed for expression and capture efficiency by measuring the response of an anti-capture tag antibody binding to the SPOC array via SPR. Individual proteins are expected to have variable expression. Low expressors may benefit from changes in the plasmid expression vector or changes in the concentration of plasmid DNA in the nanowells.

Validating protein functionality & 3D structure

Each SPOC array is equipped with a specific set of proteins for quality control (QC) purposes. In addition, SPOC array proteins are validated through a variety of mechanisms.

Antibody binding: Antigen-specific antibodies for the protein are analyzed for binding via SPR to ensure proper capture and expression on each run. Replicates of the same QC protein are present on each SPOC array to measure consistency of expression and capture across the entire array and between SPR chips (intra- and inter-assay precision). When structurally-dependent antibodies are available, they are utilized to ensure accurate 3D structure formation during protein expression. In some cases, incubation with structural enhancers during or post-expression may be required to ensure 3D structure formation.
Substrates of enzymes: Some proteins are subject to PTMs which can be performed on the SPOC chip, post-expression and capture. As an example, we demonstrated post-expression citrullination by incubating a SPOC biosensor chip with PAD2 and measuring an anti-citrullination specific antibody binding via SPR before and after incubation. The successful binding after PAD2 incubation indicated that the protein was properly folded as a substrate for the citrullination enzyme.
Enzymatic activity: Specific proteins expressed on SPOC chips have been demonstrated to exhibit enzymatic activity. As an example, we demonstrated Src protein on SPOC chip to be enzymatically and functionally active. We showed that the on array Src protein has functional enzymatic activity by de-phosphorylating and re-phosphorylating the array followed by incubation with a phospho-specific antibody and SPR analysis at each step.

SPOC arrays routinely contain soluble proteins. In the cases of transmembrane proteins, we currently express extracellular domains. Future advancements will enable full-length transmembrane proteins to be expressed and captured directly onto the SPOC chips.

References

Karthikeyan K, Barker K, Tang Y, Kahn P, Wiktor P, Brunner A, Knabben V, Takulapalli B, Buckner J, Nepom G, LaBaer J, Qiu J. A Contra Capture Protein Array Platform for Studying Post-translationally Modified (PTM) Auto-antigenomes. Mol Cell Proteomics. 2016 Jul;15(7):2324-37. doi: 10.1074/mcp.M115.057661. Epub 2016 May 2. PMID: 27141097; PMCID: PMC4937507.