determining-the-serum-stability-of-human-adenosine-deaminase-1-enzyme

Determining the Serum Stability of Human Adenosine Deaminase 1 Enzyme

The concept of enzyme stability is typically used to refer to an enzyme's thermostability - its ability to retain structure and activity as temperature ...

Georgia Institute of Technology

The full application of the diverse toolkit of protein engineering has made it easier to control the immune system. In particular, synthetic cytokine variants and engineered immune receptor platforms have shown promise for the treatment of various indications with dysregulated immune function, particularly cancer. Here, we review recent advances in the control of immune cell signaling and therapeutic potency that have employed protein engineering strategies. We further discuss how safety concerns are driving the design of immunotherapeutics toward 'user-defined' control or requiring multiple distinct inputs before a functional response, highlighting emergent control strategies employed for chimeric antigen receptor (CAR) engineering.

Protein engineering: a driving force toward synthetic immunology.

Tumors accumulate metabolites that deactivate infiltrating immune cells and polarize them toward anti-inflammatory phenotypes. We provide a comprehensive review of the complex networks orchestrated by several of the most potent immunosuppressive metabolites, highlighting the impact of adenosine, kynurenines, prostaglandin E2, and norepinephrine and epinephrine, while discussing completed and ongoing clinical efforts to curtail their impact. Retrospective analyses of clinical data have elucidated that their activity is negatively associated with prognosis in diverse cancer indications, though there is a current paucity of approved therapies that disrupt their synthesis or downstream signaling axes. We hypothesize that prior lukewarm results may be attributed to redundancies in each metabolites' synthesis or signaling pathway and highlight routes for how therapeutic development and patient stratification might proceed in the future.

Immunosuppressive metabolites in tumoral immune evasion: redundancies, clinical efforts, and pathways forward.

The eukaryotic yeast Saccharomyces cerevisiae is a model host utilized for whole cell biocatalytic conversions, protein evolution, and scientific inquiries into the pathogenesis of human disease. Over the past decade, the scale and pace of such studies has drastically increased alongside the advent of novel tools for both genome-wide studies and targeted genetic mutagenesis. In this review, we will detail past and present (e.g., CRISPR/Cas) genome-scale screening platforms, typically employed in the context of growth-based selections for improved whole cell phenotype or for mechanistic interrogations. We will further highlight recent advances that enable the rapid and often continuous evolution of biomolecules with improved function. Additionally, we will detail the corresponding advances in high throughput selection and screening strategies that are essential for assessing or isolating cellular and protein improvements. Finally, we will describe how future developments can continue to advance yeast high throughput engineering.

High throughput mutagenesis and screening for yeast engineering.

Homo sapiens adenosine deaminase isoform 1 (HsADA1) hydrolyzes adenosine and 2-deoxyadenosine as a key step in the purine nucleoside salvage pathway. Some HsADA1 mutations have severe deleterious effects, as is the case in a severe combined immunodeficiency resulting from loss of enzyme activity (ADA-SCID). Other mutations that reduce enzyme activity, for instance the Asp8Asn (D8N) variant, do not cause ADA-SCID but are correlated with other consequences to health. To ease further study of HsADA1 and its variants, we optimized an inexpensive, recombinant expression process in an Escherichia coli host through multiplexed parameter testing enabled by a lysate-based microtiter plate assay. We demonstrate the importance of gene codon usage, induction time and temperature, and alcohol supplementation towards improving enzyme yield to a final titer of 5 mg per liter of culture. We further show that use of a double-histidine-tag (his-tag) system greatly improves purity. We then utilize our expression and purification framework to produce the HsADA1 D8N variant, which had previously not been purified to homogeneity. We confirm that the D8N variant is ∼30% less active than the wildtype HsADA1 and show that it better retains its activity in human serum. Additionally, we show that both HsADA1 and the D8N variant have heightened activity in serum, driven in part by a previously undescribed phenomenon involving albumin. Therefore, this work presents a valuable process to produce HsADA1 that allows for insights into it and its variants' behavior. We also confirm the utility of lysate-based activity assays towards finding optimal E. coli expression conditions for enzymes and show how fusing his-tags in tandem can enhance product purity.

Article	Year
Protein engineering: a driving force toward synthetic immunology. Trends in biotechnology\| PubMed ID: 34627648	2022
Immunosuppressive metabolites in tumoral immune evasion: redundancies, clinical efforts, and pathways forward. Journal for immunotherapy of cancer\| PubMed ID: 34667078	2021
High throughput mutagenesis and screening for yeast engineering. Journal of biological engineering\| PubMed ID: 36575525	2022
Optimized expression and purification of a human adenosine deaminase in E. coli and characterization of its Asp8Asn variant. Protein expression and purification\| PubMed ID: 37683902	2024

John J. Blazeck

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