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Process intensification of core streptavidin production through high-cell-density cultivation of recombinant E. coli and a temperature-based refolding method

Chua LH, Tan SC, Liew MWO

J Biotechnol, 2018 Jun 20;276-277:34-41.
PMID: 29679607 DOI: 10.1016/j.jbiotec.2018.04.012

An intensified process was developed that enables high level production of recombinant core streptavidin (cSAV), a non-glycosylated tetrameric protein utilised in a wide range of applications. A pH-stat fed-batch feeding strategy was employed to achieve high-cell-density and improve volumetric yield of cSAV which was expressed as inclusion bodies (IBs). The effect of induction at different cell densities (OD 20, 60 and 100) on volumetric and specific yield were then studied. Highest volumetric yield of cSAV (1550 mg L-1) was obtained from induction at OD 100 without significant reductions in specific yield. To recover active cSAV from IBs, the possibility of refolding using a temperature-based refolding method was investigated. Refolded cSAV obtained from temperature-based refolding were then compared against cSAV refolded with conventional dialysis and dilution methods using quantitative and qualitative metrics. The temperature-based refolding method was found to improve the yield of cSAV by 6-18% in comparison to conventional methods without compromising quality. Intensification was achieved by reductions in process volumes and a more concentrated product stream. Using the newly developed process, the volumetric yield of cSAV IBs was improved by thirty-six fold in comparison to low-cell-density shake flask cultivation, and 33% of cSAV can be recovered from IBs at 90% purity.
Inhibition of Cytochrome P450s by Strobilanthes crispus Sub-Fraction (F3): Implication for Herb-Drug Interaction

Yong YF, Liew MWO, Yaacob NS

Eur J Drug Metab Pharmacokinet, 2022 Feb 11.
PMID: 35146636 DOI: 10.1007/s13318-022-00754-z

BACKGROUND AND OBJECTIVE: Strobilanthes crispus Blume sub-fraction (F3) has been reported to be cytotoxic against cancer cells and to cause murine mammary tumor regression. Potential utilization of F3 as an adjuvant in breast cancer treatment to alleviate chemotherapeutic drug resistance is currently hampered by potential cytochrome P450 (CYP)-mediated herb-drug interactions (HDIs). The current study assessed the inhibitory potency of F3 towards five CYP enzymes involved in tamoxifen metabolism.
METHODS: Potential CYP inhibition by F3 was first determined using fluorescence assays, using known CYP inhibitors as reference. To further ascertain the inhibitory potency and mode of inhibition, high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis of specific metabolites of a CYP probe substrate was conducted.
RESULTS: The half-maximal inhibitory concentration (IC50) values indicate that F3 exhibited relatively weak inhibition on CYP2B6, CYP2C19, CYP2D6, and CYP3A4. Highest susceptibility to inhibition by F3 was observed for CYP2C9, where the IC50 value from fluorescence-based assay was 35-fold higher than control. Further analysis by HPLC-MS/MS revealed relatively weak mixed-type inhibition of F3 on CYP2C9, as indicated by IC50 and inhibition constant (KI) values. The risk of clinically significant CYP2C9 inhibition by F3 was then predicted based on the attained KI value and the presumed amount of F3 absorbed from S. crispus leaves following consumption. The calculated maximum plasma concentration to inhibition constant Cmax/KI) ratio suggests that F3 consumption could potentially result in clinically significant drug interactions with medications metabolized by CYP2C9.
CONCLUSION: Taken together, the results revealed a low probability of inhibition by F3 on CYP enzymes involved in tamoxifen metabolism. However, further in vivo investigation is necessary for potential F3 interaction with CYP2C9. The utility of a preliminary in vitro approach in the assessment of potential HDI was demonstrated in this study.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method development for screening of potential tamoxifen-drug/herb interaction via in vitro cytochrome P450 inhibition assay

Yong YF, Tan SC, Liew MWO, Yaacob NS

J Chromatogr B Analyt Technol Biomed Life Sci, 2020 May 08;1148:122148.
PMID: 32416571 DOI: 10.1016/j.jchromb.2020.122148

Screening for potential drug-drug interaction (DDI) or herb-drug interaction (HDI) using in vitro cytochrome P450 inhibition (IVCI) assays requires robust analytical methods with high sensitivity and reproducibility. Utilization of liquid chromatography-mass spectrometry (LC-MS) for analyte quantification is often hampered by the presence of non-volatile IVCI sample buffer constituents that often results in ion suppression. In this study, to enable screening of drug interactions involving tamoxifen (TAM) metabolism using IVCI-LC-MS/MS, a liquid-liquid extraction (LLE) method was developed and optimized for sample clean-up. Utilization of chloroform as extraction solvent and adjustment of sample pH to 11 was found to result in satisfactory recovery (>70%) and low ion suppression (<19%). A LC-MS/MS method was subsequently developed and validated for simultaneous quantification of major TAM metabolites, such as N-desmethyltamoxifen (NDT), endoxifen (EDF) and 4-hydroxytamoxifen (HTF) to enable IVCI sample analysis. Satisfactory separation of E-/Z-isomers of endoxifen with peak resolution (Rs) of 1.9 was achieved. Accuracy and precision of the method was verified within the linear range of 0-50 ng/mL for NDT, 0-25 ng/mL for HTF and 0-25 ng/mL for EDF (E/Z isomers). Inhibitory potency (IC50, Ki and mode of inhibition) of known CYP inhibitors and Strobilanthes crispus extract was then evaluated using the validated method. In summary, the results demonstrated applicability of the developed LLE and validated LC-MS/MS method for in vitro screening of DDI and HDI involving TAM metabolism.
Evaluation of Salmonella Typhi antigen YncE alongside HlyE for the detection of typhoid fever and its carriers

Franklin F, Chong CW, Chua LH, Anthony AA, Liew MWO, Aziah I, et al.

Med Microbiol Immunol, 2020 Oct;209(5):593-601.
PMID: 32246197 DOI: 10.1007/s00430-020-00667-1

Typhoid fever is a disease caused by Salmonella Typhi that was implicated in millions of illnesses worldwide annually. Individuals that do not recover fully from typhoid fever can become asymptomatic carriers of the disease. Host antibodies against the S. Typhi antigens, HlyE (for acute typhoid) and YncE (for carriers) were previously reported to be useful biomarkers for the disease. Here, we expressed and purified recombinant HlyE and YncE antigens and tested the IgG, IgA and IgM responses in 422 sera samples retrieved from acute typhoid patients, other febrile, food handlers, and healthy individuals. The results showed that HlyE-IgG, -IgA and -IgM ELISAs have a collective sensitivity of 83% while YncE-IgG and -IgA ELISAs identified 16 possible carriers based on their antibody profiles. The identification of sensitive biomarkers for typhoid carrier detection is crucial for disease eradication.
Intensification of Inclusion Body Processing via Temperature-Based Refolding

Wong RS, Alias NNM, Ong EBB, Liew MWO

Methods Mol Biol, 2023;2617:189-200.
PMID: 36656525 DOI: 10.1007/978-1-0716-2930-7_13

Inclusion bodies (IB) are dense insoluble aggregates of mostly misfolded polypeptides that usually result from recombinant protein overexpression. IB formation has been observed in protein expression systems such as E. coli, yeast, and higher eukaryotes. To recover soluble recombinant proteins in their native state, IB are commonly first solubilized with a high concentration of denaturant. This is followed by concurrent denaturant removal or reduction and a transition into a refolding-favorable chemical environment to facilitate the refolding of solubilized protein to its native state. Due to the high concentration of denaturant used, conventional refolding approaches can result in dilute products and are buffer inefficient. To circumvent the limitations of conventional refolding approaches, a temperature-based refolding approach which combines a low concentration of denaturant (0.5 M guanidine hydrochloride, GdnHCl) with a high temperature (95 °C) during solubilization was proposed. In this chapter, we describe a temperature-based refolding approach for the recovery of core streptavidin (cSAV) from IB. Through the temperature-based approach, intensification was achieved through the elimination of a concentration step which would be required by a dilution approach and through a reduction in buffer volumes required for dilution or denaturant removal. High-temperature treatment during solubilization may have also resulted in the denaturation and aggregation of undesired host-cell proteins, which could then be removed through a centrifugation step resulting in refolded cSAV of high purity without the need for column purification. Refolded cSAV was characterized by biotin-binding assay and SDS-PAGE, while purity was determined by RP-HPLC.
Fulltext Cytotoxin-mediated silk gland organ dysfunction diverts resources to enhance silkworm fecundity by potentiating nutrient-sensing IIS/TOR pathways

Lye PY, Shiraki C, Fukushima Y, Takaki K, Liew MWO, Yamamoto M, et al.

iScience, 2024 Feb 16;27(2):108853.
PMID: 38303707 DOI: 10.1016/j.isci.2024.108853

Energy reserves, primarily stored in the insect's fat body, are essential for physiological processes such as reproduction and cocoon formation. However, whether these processes are mutually constraining is unknown. Here, we showed that cocoon-free silkworms accumulate amino acid constituents of silk proteins in the hemolymph and maintain lipid and sugar reserves in the pupal fat body by repressing the expression of sericin and fibroin genes in the middle and posterior silk glands, respectively, via butterfly pierisin-1A catalytic domain expression. This, in turn, upregulates insulin/insulin-like signaling and target of rapamycin (IIS/TOR) signaling, which enhances vitellogenesis and accelerates ovarian development, thus contributing to increased fecundity. The impacts of semi-starvation on fecundity and egg hatchability were also less pronounced in cocoon-free silkworms compared with wildtype silkworms. These data uncover the resource allocation trade-off between cocoon formation and fecundity and demonstrate that nutritional signaling plays a role in regulating silkworm reproduction.