A map of the rubisco biochemical landscape.

Rubisco is the primary CO2 fixing enzyme of the biosphere yet has slow kinetics. The roles of evolution and chemical mechanism in constraining the sequence landscape of rubisco remain debated. In order to map sequence to function, we developed a massively parallel assay for rubisco using an engineered E. coli where enzyme function is coupled to growth. By assaying >99% of single amino acid mutants across CO2 concentrations, we inferred enzyme velocity and CO2 affinity for thousands of substitutions. We identified many highly conserved positions that tolerate mutation and rare mutations that improve CO2 affinity. These data suggest that non-trivial kinetic improvements are readily accessible and provide a comprehensive sequence-to-function mapping for enzyme engineering efforts.

Development of a Cross-Reactive Monoclonal Antibody for Detecting the Tumor Stroma.

Here, we document the discovery of a monoclonal antibody that selectively binds to both human and murine fibroblast activation protein alpha (FAP), a serine protease that is overexpressed on cancer-associated fibroblasts (CAFs), making it an attractive therapeutic target for the aiding and abetting tumor microenvironment. The lead antibody, B12, was identified from a naïve murine single-chain variable fragment antibody phage display library screened against recombinant human FAP on magnetic beads. The heavy and light chains of B12 were cloned into full-length human immunoglobulin 1 (IgG) vectors and expressed as a chimeric monoclonal antibody (B12 IgG). We engineered a drug-resistant prostate cancer cell line, CWR-R1-EnzR, to express human FAP for antibody characterization and validation (R1-EnzRFAP). B12 IgG selectively bound to the R1-EnzRFAP cells by flow cytometry and was internalized in vitro by confocal microscopy. B12 IgG was further evaluated as a near-infrared (NIR) optical imaging probe in R1-EnzRFAP and parental xenograft models. High tumor uptake and retention of the NIR probe was observed in the R1-EnzRFAP xenografts, and endogenous expression of murine stromal origin FAP was detected in the parental xenografts. Ex vivo evaluation of these models by immunohistochemistry documented B12 IgG localization to both human and murine FAP-expressing cells.