Advancing large-scale production of TEV protease through an innovative NT* tag-based fusion construct.

Tobacco etch virus Protease (TEVp), a cysteine protease, is renowned for its remarkable specific proteolysis, making it an invaluable tool for removing fusion tags from recombinant proteins. However, TEV protease's inherent insolubility limits its broad application. Fusion constructs like an N-terminal MBP fusion, known for its improved solubility, have been employed for TEVp production to address this issue. In this study, we fused the TEVp with the N-terminal domain of the spider silk protein, specifically utilizing a charge-reversed mutant (D40K/K65D) of the N-terminal domain of major ampullate spidroin-1 protein from Euprosthenops australis, referred to as NT*. This fusion construct contains a TEVp cleavage site, enabling intracellular self-processing and the release of a His7-tagged protease. The significant increase in soluble protein expression allowed us to purify approximately 90-100 mg of TEVp from a 1-L E. coli culture, surpassing previous findings by a considerable margin. The enzyme remained stable and catalytically active even after several months of storage in a deep freezer (-80 °C).

Functional manipulation of a calcium-binding protein from Entamoeba histolytica guided by paramagnetic NMR.

EhCaBP1, one of the calcium-binding proteins from Entamoeba histolytica, is a two-domain EF-hand protein. The two domains of EhCaBP1 are structurally and functionally different from each other. However, both domains are required for structural stability and a full range of functional diversity. Analysis of sequence and structure of EhCaBP1 and other CaBPs indicates that the C-terminal domain of EhCaBP1 possesses a unique structure compared with other family members. This had been attributed to the absence of a Phe-Phe interaction between highly conserved Phe residues at the -4 position in EF-hand III (F[-4]; Tyr(81)) and at the 13th position in EF-hand IV (F[+13]; Phe(129)) of the C-terminal domain. Against this backdrop, we mutated the Tyr residue at the -4th position of EF III to the Phe residue (Y81F), to bring in the Phe-Phe interaction and understand the nature of structural and functional changes in the protein by NMR spectroscopy, molecular dynamics (MD) simulation, isothermal titration calorimetry (ITC), and biological assays, such as imaging and actin binding. The Y81F mutation in EhCaBP1 resulted in a more compact structure for the C-terminal domain of the mutant as in the case of calmodulin and troponin C. The compact structure is favored by the presence of a π-π interaction between Phe(81) and Phe(129) along with several hydrophobic interactions of Phe(81), which are not seen in the wild-type protein. Furthermore, the biological assays reveal preferential membrane localization of the mutant, loss of its colocalization with actin in the phagocytic cups, whereas retaining its ability to bind G- and F-actin.