Optimized expression and purification of a human adenosine deaminase in E. coli and characterization of its Asp8Asn variant

腺苷脱氨酶 生物化学 大肠杆菌 生物 重组DNA 基因 分子生物学 腺苷
作者
Maria Rain Jennings,S.J. Min,Grace S. Xu,Kassandra Homayuni,Bhavana Suresh,Yusef Haikal,John Blazeck
出处
期刊:Protein Expression and Purification [Elsevier BV]
卷期号:213: 106362-106362 被引量:1
标识
DOI:10.1016/j.pep.2023.106362
摘要

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.
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