Expression of scFv-anti-CHIKV-E2 in Escherichia coli with chaperones Co-expression, and its functional assay by electrochemical immunosensor.

Single Chain Variable Fragment (scFv), a small fragment of antibody can be used to substitute the monoclonal antibody for diagnostic purposes. Production of scFv in Escherichia coli host has been a challenge due to the potential miss-folding and formation of inclusion bodies. This study aimed to express anti-CHIKV E2 scFv which previously designed specifically for Asian strains by co-expression of three chaperones that play a role in increasing protein solubility; GroEL, GroES, and Trigger Factor. The scFv and chaperones were expressed in Origami B E. coli host under the control of the T7 promoter, and purified using a Ni-NTA column. Functional assay of anti-CHIKV-E2 scFv was examined by electrochemical immunosensor using gold modified Screen Printed Carbon Electrode (SPCE), and characterized by differential pulses voltammetry (DPV) using K3[Fe(CN)6] redox system and scanning microscope electron (SEM). The experimental condition was optimized using the Box-Behnken design. The results showed that co-expression of chaperone increased the soluble scFv yield from 54.405 µg/mL to 220.097 µg/mL (~5×). Furthermore, scFv can be used to detect CHIKV-E2 in immunosensor electrochemistry with a detection limit of 0.74048 ng/mL and a quantification limit of 2,24388 ng/mL. Thus, the scFv-anti-CHIKV-E2 can be applied as a bioreceptor in another immunoassay method.

Theoretical and Experimental Studies on the Evidence of 1,3-ß-Glucan in Marennine of Haslea ostrearia.

Marennine, a blue pigment produced by the blue diatom Haslea ostrearia, is known to have some biological activities. This pigment is responsible for the greening of oysters on the West Coast of France. Other new species of blue diatom, H. karadagensis, H. silbo sp. inedit., H. provincialis sp. inedit, and H. nusantara, also produce marennine-like pigments with similar biological activities. Aside from being a potential source of natural blue pigments, H. ostrearia-like diatoms present a commercial potential for the aquaculture, food, cosmetics, and health industries. Unfortunately, for a hundred years, the exact molecular structure of this bioactive compound has remained a mystery. A lot of hypotheses regarding the chemical structure of marennine have been proposed. The recent discovery of this structure revealed that it is a macromolecule, mainly carbohydrates, with a complex composition. In this study, some glycoside hydrolases were used to digest marennine, and the products were further analyzed using nuclear magnetic resonance (NMR) and mass spectroscopy (MS). The reducing sugar assay showed that marennine was hydrolyzed only by endo-1,3-ß-glucanase. Further insight into the structure of marennine was provided by the spectrum of 1H NMR, MS, a colorimetric assay, and a computational study, which suggest that the chemical structure of marennine contains 1,3-ß-glucan.

Residual Interactions of LL-37 with POPC and POPE:POPG Bilayer Model Studied by All-Atom Molecular Dynamics Simulation.

LL-37 is a membrane-active antimicrobial peptide (AMP) that could disrupt the integrity of bacterial membranes due to its inherent cationic and amphipathic nature. Developing a shorter derivative of a long peptide such as LL-37 is of great interest, as it can reduce production costs and cytotoxicity. However, more detailed information about the residual interaction between LL-37 and the membrane is required for further optimization. Previously, molecular dynamics simulation using mixed all-atom and united-atom force fields showed that LL-37 could penetrate the bilayer membrane. This study aimed to perform all-atom molecular dynamics simulations, highlighting the residual interaction of LL-37 with the simplest model of the bacterial membrane, POPE:POPG (2:1), and compare its interaction with the POPC, which represents the eukaryotic membrane. The result showed leucine-leucine as the leading residues of LL-37 that first contact the membrane surface. Then, the cationic peptide of LL-37 started to penetrate the membrane by developing salt bridges between positively charged amino acids, Lys-Arg, and the exposed phosphate group of POPE:POPG, which is shielded in POPC. Residues 18 to 29 are suggested as the core region of LL-37, as they actively interact with the POPE:POPG membrane, not POPC. These results could provide a basis for modifying the amino acid sequence of LL-37 and developing a more efficient design for LL-37 derivatives.

Expression, Sarkosyl Solubilization, DNase Activity, Purification, and SPR Binding Affinity of Recombinant Diphtheria Toxoid (rCRM197EK) Expressed in Escherichia coli BL21(DE3).

CRM197EK is a derivative of diphtheria toxoid cross-reactive material-197 (CRM197) with two-point mutations (K51E and E148K) to improve its properties for a vaccine conjugate and drug delivery. A previous study has shown that intracellularly expressing CRM197EK in Escherichia coli (E. coli) host formed inclusion bodies that need a complicated purification and refolding step. Protein purification from inclusion bodies can be overcome by solubilization of inclusion bodies by using N-lauroyl sarcosine (sarkosyl). In this work, recombinant CRM197EK (rCRM197EK) was expressed in E. coli BL21 (DE3) as inclusion bodies, then solubilized using sarkosyl to form a soluble rCRM197EK without the need for a renaturation process. Furthermore, rCRM197EK was purified using the Ni-NTA column, characterized by SDS-PAGE and Western Blot, and its biological activity was assayed through its DNase activity. Moreover, its binding affinity with anti-diphtheria toxin (DT) antibody was measured using the surface plasmon resonance (SPR). The result showed that solubilization with sarkosyl form soluble rCRM197EK (61.61 kDa) was confirmed by SDS-PAGE and Western Blot with a yield of 2.8 mg/mL. rCRM197EK shows DNase activity, and the SPR assay shows that it can interact with an anti-DT antibody with a binding energy of - 9.2 kcal/mol.

Enzymatic Performance of Aspergillus oryzae α-Amylase in the Presence of Organic Solvents: Activity, Stability, and Bioinformatic Studies.

Enzymatic reactions can be modulated by the incorporation of organic solvents, leading to alterations in enzyme stability, activity, and reaction rates. These solvents create a favorable microenvironment that enables hydrophobic reactions, facilities enzyme-substrate complex formation, and reduces undesirable water-dependent side reactions. However, it is crucial to understand the impact of organic solvents on enzymatic activity, as they can also induce enzyme inactivation. In this study, the enzymatic performance of Aspergillus oryzae α-amylase (Taka-amylase) in various organic solvents both experimentally and computationally was investigated. The results demonstrated that ethanol and ether sustain Taka-amylase activity up to 20% to 25% of the organic solvents, with ether providing twice the stability of ethanol. Molecular dynamics simulations further revealed that Taka-amylase has a more stable structure in ether and ethanol relative to other organic solvents. In addition, the analysis showed that the loop located near the active site in the AB-domain is a vulnerable site for enzyme destabilization when exposed to organic solvents. The ability of Taka-amylase to preserve the secondary loop structure in ether and ethanol contributed to the enzyme's activity. In addition, the solvent accessibility surface area of Taka-amylase is distributed throughout all enzyme structures, thereby contributing to the instability of Taka-amylase in the presence of most organic solvents.