Proteobacterial Antimicrobial Compound Efflux (PACE) family proteins are one of seven types of multidrug efflux pumps in Gram-negative bacteria. PACE proteins can actively efflux synthetic biocides, including the antiseptic chlorhexidine, whilst their physiological substrates are polyamines. PACE proteins contain four putative transmembrane-spanning alpha-helices, and experimental evidence suggests that they mainly exist and function in a dimeric state, but the high-resolution structural organisation and molecular mechanism of PACE proteins are yet to be elucidated experimentally. As an essential first step to achieve this, we employed a strategy for gene cloning, expression screening and large-scale purification of representative PACE proteins. The genes of 24 proteins were successfully cloned into IPTG-inducible plasmid pTTQ18 directly upstream from a His6-tag coding sequence and transformed into E. coli BL21(DE3) cells. Small-scale expression tests identified seven proteins amplified at a sufficient level for larger-scale cultures and purification. Based on results from 30-litre fermentor cultures and inner membrane preparations, four proteins (A1S_2063, Fbal_3166, STY_3166, Tmarg_opt) representing distinct phylogenetic groups of the PACE family, were progressed to detergent solubilisation and purification. These proteins had purities of 86, 84, 80 and 78%, and purification yields of 1.1, 1.1, 1.3 and 1.0 mg/litre cell culture, respectively. The detergent-solubilised purified proteins had far-UV circular dichroism spectra consistent with alpha-helical secondary structure, producing melting temperatures of 46.7, 34.2, 32.6 and 37.6 C, respectively. A1S_2063 was most stable and might be best for structure elucidation. Secondary structure in all purified proteins appeared reasonably stable for performing biochemical and biophysical experiments up to 25 C.

Background: Non-small cell lung cancer (NSCLC) represents nearly 85% of lung cancer cases globally and remains a major cause of cancer mortality. Although immune checkpoint inhibitors improve survival, durable responses occur in a limited number of patients, highlighting the urgent need for reliable, minimally invasive predictive biomarkers. Objective: To evaluate whether specific serum protein signatures can predict clinical response to immune checkpoint inhibitors in patients with NSCLC. Methods: This prospective cohort study was conducted at National Institute of Cancer Research and Hospital, Dhaka, Bangladesh from January 2024 to December 2024. A total of 67 histologically confirmed NSCLC patients receiving ICI therapy were enrolled using purposive sampling. Baseline serum samples were obtained before treatment and analyzed for selected protein biomarkers related to immune regulation and inflammation. Clinical response was evaluated at 12 weeks using the RECIST criteria. Data were processed in SPSS 23.0, applying logistic regression and ROC curve analyses. Results: Of the 67 patients, 29 (43.3%) achieved partial response or stable disease, whereas 38 (56.7%) experienced disease progression. Higher baseline pro-inflammatory protein levels were significantly associated with poor response (p<0.05), while elevated immune-activating proteins predicted favorable outcomes (p<0.01). The combined serum protein signature showed strong predictive accuracy (AUC=0.81; 95% CI: 0.70–0.91). Conclusion: Distinct serum protein signatures may serve as non-invasive predictive biomarkers for clinical response to ICIs in NSCLC patients. These findings support further validation in larger, multicenter studies to optimize personalized immunotherapy strategies.