Evaluating the ability of different chaperones in improving soluble expression of a triple-mutated human interferon gamma in Escherichia coli.

Human interferon gamma (hIFN-γ) plays a pivotal role as a soluble cytokine with diverse functions in both innate and adaptive immunity. In a previous investigation, we pinpointed three critical amino acid residues, i.e., threonine (T) 27, phenylalanine (F) 29, and leucine (L) 30, on the IFN-γ structure, which are integral to the epitope recognized by anti-IFN-γ autoantibodies. It is crucial to impede the interaction between this epitope and autoantibodies for effective therapy in adult-onset immunodeficiency (AOID). However, the challenge arises from the diminished solubility of the T27AF29L30A mutant in Escherichia coli BL21(DE3). This study delves into a targeted strategy aimed at improving the soluble expression of IFN-γ T27AF29AL30A. This is achieved through the utilization of five chaperone plasmids: pG-KJE8, pKJE7, pGro7, pG-Tf2, and pTf16. These plasmids, encoding cytoplasmic chaperones, are co-expressed with the IFN-γ mutant in E. coli BL21(DE3), and we meticulously analyze the proteins in cell lysate and inclusion bodies using SDS-PAGE and Western blotting. Our findings reveal the remarkable efficacy of pG-KJE8, which houses cytoplasmic chaperones DnaK-DnaJ-GrpE and GroEL-GroES, in significantly enhancing the solubility of IFN-γ T27AF29AL30A. Importantly, this co-expression not only addresses solubility concerns but also preserves the functional dimerized structure, as confirmed by sandwich ELISA. This promising outcome signifies a significant step forward in developing biologic strategies for AOID.

Characterization of Bacterial Cellulose Produced by Komagataeibacter maltaceti P285 Isolated from Contaminated Honey Wine.

Bacterial cellulose (BC), a biopolymer, is synthesized by BC-producing bacteria. Almost all producing strains are classified in the family Acetobacteraceae. In this study, bacterial strain P285 was isolated from contaminated honey wine in a honey factory in northern Thailand. Based on 16S rRNA gene sequence identification, the strain P285 revealed 99.8% identity with Komagataeibacter maltaceti LMG 1529 T. K. maltaceti P285 produced the maximum BC production at 20-30 °C and an initial media pH of 9.0. The highest BC production in modified mineral salt medium (MSM) was exhibited when glucose (16%, w/v) and yeast extract (3.2%, w/v) were applied as carbon and nitrogen sources, respectively. When sugarcane (8-16%, w/v) or honey (ratio of honey to water = 1: 4) supplemented with yeast extract was used, the BC production was greater. The characterization of BC synthesized by K. maltaceti P285 was undertaken using scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectrometry. Meanwhile, X-ray diffraction results confirmed the presence of crystalline cellulose (2θ = 18.330, 21.390 and 22.640°). The maximum temperature of BC degradation was observed at 314 °C. Tensile properties analysis of hydrated and dried BC showed breaking strength of 1.49 and 0.66 MPa, respectively. These results demonstrated that K. maltaceti P285 has a high potential for BC production especially when grown in high initial media pH. Therefore, the strain would be suitable as an agent to make BC, the value-added product in the related factories.