L-threonine is an essential amino acid required for mucin synthesis and intestinal barrier maintenance. The gut commensal Akkermansia muciniphila, which resides in the mucus layer, encodes a putative L-threonine 3-dehydrogenase (Am_TDH) whose biochemical properties remain unknown. Here, we report the heterologous expression, purification, and biochemical characterization of this enzyme. The enzyme was characterized as a member of the zinc-containing medium-chain dehydrogenase/reductase (MDR) superfamily and shown to form a homotetramer with a molecular mass of 154 kDa. Am_TDH was strictly NAD+-dependent, exhibiting maximal specific activity toward L-threonine (30.3 ± 0.4 U mg−1). Optimal catalytic activity was observed at pH 8.5 and 37 °C. The Km values for L-threonine and NAD+ at 37 °C were 3.56 ± 0.48 mM and 0.65 ± 0.19 mM, respectively. These findings reveal a physiologically adapted, stereospecific TDH and provide molecular insights into amino acid metabolism by a gut symbiont.
Increasing the yield of recombinant proteins can save time and materials and, consequently, any strategy that seems promising for achieving this objective is worth investigating. Lately, it has been proposed that modifications of upstream sequences from pET vectors would enhance the transcription efficiency. To validate this approach, we expressed a pool of constructs in modified pET vectors and compared the yields with those obtained using unmodified pET-derived vectors. Constructs were selected according to their different structural properties and protein yields. In our experimental setting, the results were generally deceiving. However, improvements were observed when additional elements of the construct were optimized concurrently. In particular, repositioning the His-tag from the C-terminus to the N-terminus positively affected the final protein yield. Taken together, these findings indicate that the modified vectors developed in this study represent one of several factors to consider during the initial screening aimed at identifying optimal expression conditions for a new construct, rather than a broadly applicable solution for enhancing the yield of any protein construct.
The rapid emergence of antimicrobial resistance has intensified the demand for novel anti-infective agents, positioning antimicrobial peptides (AMPs) as promising therapeutic alternatives. However, large scale production of AMPs remains challenging due to their small size, cationic nature, host toxicity, and high cost of chemical synthesis. In this study, we report an efficient and economically viable intein-mediated recombinant bioprocess for the production of the short antimicrobial peptide Lasioglossin-III (LL-III) in its C-terminally amidated form using Escherichia coli-based expression platform. A codon-optimized LL-III gene was fused to the Mxe GyrA intein along with a chitin binding domain (CBD) using the pTWIN1 expression system, enabling stable expression, affinity purification, and controlled auto-cleavage. The fusion protein was expressed in soluble form at low temperature and purified using chitin resin under optimized binding conditions. Notably, the chitin resin retained its binding capacity across multiple reuse cycles, improving the overall process-economics; auto-cleavage under mild reducing conditions released the mature peptide, which was confirmed by RP-HPLC and LC/MS to be identical to chemically synthesized standard peptide. The optimized process yielded ∼4.4 mg L−1 of recombinant peptide leading to significant reduction in production costs at the current cleavage efficiency. Overall, this study establishes a scalable, environmentally sustainable, and cost-effective bioprocessing platform not only for short AMPs, but also for other peptide-based therapeutics as well.
Tuberculosis remains among the top ten deadliest infectious diseases worldwide, primarily due to delayed diagnosis, co-infection with HIV, and multidrug resistance. Targeting early secreted mycobacterial antigens, including MPT64, ESAT6, and CFP10, may be a viable strategy for detecting the onset of tuberculosis. Although recombinant MPT64 has been produced previously, limited information is available regarding its structural integrity and thermodynamic unfolding behaviour following recovery from inclusion bodies. In this study, MPT64 protein was overexpressed, affinity-purified, and characterised using biophysical and biochemical methods. The purified recombinant MPT64 had a purity greater than 95%, yielding 2 mg from 1 L of bacterial culture. Western blot analysis confirmed MPT64 identity, while ELISA demonstrated antibody recognition of refolded MPT64, with markedly reduced binding following heat denaturation, indicating that refolding preserved conformational epitopes. Far-UV circular dichroism (CD) and intrinsic fluorescence spectroscopy spectral profiles confirmed that the purified MPT64 protein retained its folded secondary and tertiary structural integrity. Thermodynamic analysis by fluorescence and Far-UV CD yielded stability values (ΔG°) of 19.9 and 26.2 kJ mol−1, with unfolding midpoints of 5.3 and 5.5 M urea, respectively. Differential scanning calorimetry and thermal circular dichroism indicated melting transitions between 62.5 and 58 °C, supporting moderate thermal stability. Collectively, these complementary measurements provide a detailed biophysical assessment of the conformational and thermal stability of recombinant MPT64 following inclusion-body recovery and refolding. The findings provide a biophysical basis for further investigation of MPT64 as a recombinant antigen for tuberculosis diagnostics.
