Modeled Hepatic/Plasma Exposures of Omeprazole Prescribed Alone in Cytochrome P450 2C19 Poor Metabolizers Are Likely Associated with Hepatic Toxicity Reported in a Japanese Adverse Event Database.

Omeprazole, a gastric acid pump inhibitor, is repeatedly administered and is oxidatively metabolized mainly by polymorphic cytochrome P450 2C19. The prescribed dosage of omeprazole was discontinued or reduced in 47 of the 135 patients who received omeprazole alone in this survey, as recorded in the Japanese Adverse Drug Event Report database. The days to onset of omeprazole-related disorders were 3-4 d (median) and 16 d for intravenous 20-40 mg and oral 20 mg daily doses, respectively, in 34 patients for whom relevant data were available. The maximum plasma concentration of omeprazole was pharmacokinetically modeled after a single oral 40-mg dose in P450 2C19-defective poor metabolizers and was 2.4-fold higher than that in extensive metabolizers. The modeled area under the hepatic concentration curves of omeprazole in P450 2C19 poor metabolizers after virtual daily 40-mg doses for 7 d was 5.2-fold higher than that in the extensive metabolizers. Omeprazole-induced P450 2C19 (approx. 2-fold), resulting in increased hepatic intrinsic clearance in repeated doses, was considered after the second day. Virtual plasma/hepatic exposure estimated using pharmacokinetic modeling in subjects with P450 2C19 poor metabolizers indicated that these exposure levels virtually estimated could be one of causal factors for unexpected hepatic disorders induced by prescribed omeprazole, such as those resulting from drug interactions with repeatedly co-administered medicines.

CUREs for high-level Galectin-3 expression.

Galectins are a large and diverse protein family defined by the presence of a carbohydrate recognition domain (CRD) that binds ß-galactosides. They play important roles in early development, tissue regeneration, immune homeostasis, pathogen recognition, and cancer. In many cases, studies that examine galectin biology and the effect of manipulating galectins are aided by, or require the ability to express and purify, specific members of the galectin family. In many cases, E. coli is employed as a heterologous expression system, and galectin expression is induced with isopropyl ß-galactoside (IPTG). Here, we show that galectin-3 recognizes IPTG with micromolar affinity and that as IPTG induces expression, newly synthesized galectin can bind and sequester cytosolic IPTG, potentially repressing further expression. To circumvent this putative inhibitory feedback loop, we utilized an autoinduction protocol that lacks IPTG, leading to significantly increased yields of galectin-3. Much of this work was done within the context of a course-based undergraduate research experience, indicating the ease and reproducibility of the resulting expression and purification protocols.

Expression of soluble moloney murine leukemia virus-reverse transcriptase in Escherichia coli BL21 star (DE3) using autoinduction system.

Autoinduction systems in Escherichia coli can control the production of proteins without the addition of a particular inducer. In the present study, we optimized the heterologous expression of Moloney Murine Leukemia Virus derived Reverse Transcriptase (MMLV-RT) in E. coli. Among 4 autoinduction media, media Imperial College resulted the highest MMLV-RT overexpression in E. coli BL21 Star (DE3) with incubation time 96 h. The enzyme was produced most optimum in soluble fraction of lysate cells. The MMLV-RT was then purified using the Immobilized Metal Affinity Chromatography method and had specific activity of 629.4 U/mg. The system resulted lower specific activity and longer incubation of the enzyme than a classical Isopropyl ß-D-1-thiogalactopyranoside (IPTG)-induction system. However, the autoinduction resulted higher yield of the enzyme than the conventional induction (27.8%). Techno Economic Analysis revealed that this method could produce MMLV-RT using autoinduction at half the cost of MMLV-RT production by IPTG-induction. Bioprocessing techniques are necessary to conduct to obtain higher quality of MMLV-RT under autoinduction system.

Nonlinear Pharmacokinetics of Topical Flurbiprofen Gel in a Phase I Study Among Chinese Healthy Adults.

INTRODUCTION: PDX-02 (Flurbiprofen sodium) is a topical nonsteroidal anti-inflammatory drug in gel formulation for local analgesia and anti-inflammation. A Phase I clinical trial was conducted to assess the safety, tolerability, and pharmacokinetics of single and multiple doses of PDX-02 gel in Chinese healthy adults. METHODS: The trial comprised three parts: (1) a single-dose ascending study with three dose levels (0.5%, 1% to 2% PDX-02 gel) applied on a 136 cm2 skin area; (2) a multiple-dose study with either 1% or 2% PDX-02 gel applied on a 136 cm2 skin area for 7 consecutive days; and (3) a high dose group with 2% PDX-02 gel on an 816 cm2 skin area and a frequent multiple dose group with 2% PDX-02 gel on a 272 cm2 skin area four times a day for 7 consecutive days. The safety, tolerability and pharmacokinetics of the PDX-02 gel were evaluated in each part. RESULTS: A total of sixty participants completed the trial, with all adverse events recovered and all positive skin reaction being transient and recovered. The overall absorption of topical PDX-02 gel was slow with a mean peak time exceeding 9 h. The elimination rate remained consistent between dose groups. A less-than-dose-proportional nonlinear pharmacokinetics relationship was observed within the studied dose range, and this is likely due to the autoinduction of skin first-pass metabolism. CONCLUSION: The topical PDX-02 gel showed favorable safety and tolerability in both single and multiple dosing studies, with a less-than-dose-proportional nonlinear pharmacokinetics observed.

Production of reverse transcriptase from Moloney murine Leukemia virus in Escherichia coli expression system.

Reverse transcriptase (RT) is one of the most important enzymes used in molecular biology applications, enabling the conversion of RNA into complementary DNA (cDNA) that is used in reverse transcription-polymerase chain reaction (RT-PCR). The high demand of RT enzymes in biotechnological applications making the production optimization of RT is crucial for meeting the growing demand in industrial settings. Conventionally, the expression of recombinant RT is T7-induced promoter using IPTG in Escherichia coli expression systems, which is not cost-efficient. Here, we successfully made an alternative procedure for RT expression from Moloney murine leukemia virus (M-MLV) using autoinduction method in chemically defined medium. The optimization of carbon source composition (glucose, lactose, and glycerol) was analyzed using Response Surface Methodology (RSM). M-MLV RT was purified for further investigation on its activity. A total of 32.8 mg/L purified M-MLV RT was successfully obtained when glucose, glycerol, and lactose were present at concentration of 0.06%, 0.9%, and 0.5% respectively, making a 3.9-fold improvement in protein yield. In addition, the protein was produced in its active form by displaying 7462.50 U/mg of specific activity. This study provides the first step of small-scale procedures of M-MLV RT production that make it a cost-effective and industrially applicable strategy.

Engineering Escherichia coli for cost-effective production of medium-chain fatty acids from soy whey using an optimized galactose-based autoinduction system.

Medium-chain fatty acids (MCFAs) are essential chemical feedstocks. Microbial production of MCFAs offers an attractive alternative to conventional methods, but the costly media and external inducers limit its practical application. To address this issue and make MCFA production more cost-effective, an E.coli platform was developed using soy whey as a medium and galactose as an autoinducer. We first designed an efficient, stringent, homogeneous, and robust galactose-based autoinduction system for the expression of pathway enzymes by rationally engineering the promoter of the galactose-proton symporter (GalP). Subsequently, the intracellular acetyl-CoA availability and NADH regeneration were enhanced to improve the reversal of the ß-oxidation cycle. The resulting strain yielded 8.20 g/L and 16.42 g/L MCFA in pH-controlled batch fermentation and fed-batch fermentation with glucose added using soy whey as medium, respectively. This study provided a cost-effective and promising platform for MCFA production, as well as future strain development for other value-added chemicals production.

Expression, purification, crystallization of a ZIP metal transporter from Bordetella bronchiseptica (BbZIP).

Structural studies of the ZIPs have greatly improved the understanding of the working mechanism for this functionally important metal transporter family. In this chapter, we describe the procedures to overexpress, purify, and crystallize a representative bacterial ZIP from Bordetella bronchiseptica (BbZIP), the structure of which was the first one that revealed the common structural framework of the transmembrane domain conserved within the entire ZIP family. We also discuss the considerations when we designed these experiments and compare the approaches used in this study with those commonly used in other works. The protocols provided in this chapter will facilitate structural and biochemical studies of other members of the ZIP family.

Intranasal delivery of mitochondrial protein humanin rescues cell death and promotes mitochondrial function in Parkinson's disease.

Rationale: Mitochondrial dysfunction is a key factor in the pathogenesis of Parkinson's disease (PD). Accordingly, many aspects of mitochondrial function have been studied as a putative therapeutic target. Here we present a novel strategy to promote mitochondrial function and protect against Parkinson's disease by the peptide encoded within mitochondrial genome, mitochondria-derived peptide (MDP) humanin (HN). Methods: To test humanin as a potential biomarker in PD, we measured protein levels of circulating humanin from the plasma of PD patients and transgenic or neurotoxic mouse models of PD. Next, we aimed to identify whether HN peptide treatment can regulate its activity or expression. Using mouse models of PD, we assessed HN delivery to the brain via the nasal route of administration. We further revealed a possible mechanism underlying the therapeutic effectiveness of HN peptide for PD using in vitro and ex vivo model of PD. Results: Although the expression of intracellular HN was not correlated with PD, HN treatment itself could induce intracellular HN expression and enhance mitochondrial biogenesis inducing mitochondrial gene expression. After intranasal administration, HN peptide resulted in neuroprotection and behavioral recovery in an animal model of PD. Interestingly, HN peptide following intranasal delivery was found within the brain, mainly via the trigeminal pathways. Mechanistically, HN treatment induced activation of phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) signaling pathway which led to enhanced mitochondrial biogenesis resulting in upregulation of mitochondrial gene including humanin. Conclusion: These data support a novel role of mitochondrial protein humanin in mitochondrial function and neuronal survival against Parkinson's disease, in which humanin treatment is sufficient for stimulating mitochondrial gene expression.

A Mesorhizobium japonicum quorum sensing circuit that involves three linked genes and an unusual acyl-homoserine lactone signal.

Members of the genus Mesorhizobium, which are core components of the rhizosphere and specific symbionts of legume plants, possess genes for acyl-homoserine lactone (AHL) quorum sensing (QS). Here we show Mesorhizobium japonicum MAFF 303099 (formerly M. loti) synthesizes and responds to N-[(2E, 4E)-2,4-dodecadienoyl] homoserine lactone (2E, 4E-C12:2-HSL). We show that the 2E, 4E-C12:2-HSL QS circuit involves one of four luxR-luxI-type genes found in the sequenced genome of MAFF 303099. We refer to this circuit, which appears to be conserved among Mesorhizobium species, as R1-I1. We show that two other Mesorhizobium strains also produce 2E, 4E-C12:2-HSL. The 2E, 4E-C12:2-HSL is unique among known AHLs in its arrangement of two trans double bonds. The R1 response to 2E, 4E-C12:2-HSL is extremely selective in comparison with other LuxR homologs, and the trans double bonds appear critical for R1 signal recognition. Most well-studied LuxI-like proteins use S-adenosylmethionine and an acyl-acyl carrier protein as substrates for synthesis of AHLs. Others that form a subgroup of LuxI-type proteins use acyl-coenzyme A substrates rather than acyl-acyl carrier proteins. I1 clusters with the acyl-coenzyme A-type AHL synthases. We show that a gene linked to the I1 AHL synthase is involved in the production of the QS signal. The discovery of the unique I1 product enforces the view that further study of acyl-coenzyme A-dependent LuxI homologs will expand our knowledge of AHL diversity. The involvement of an additional enzyme in AHL generation leads us to consider this system a three-component QS circuit. IMPORTANCE We report a Mesorhizobium japonicum quorum sensing (QS) system involving a novel acyl-homoserine lactone (AHL) signal. This system is known to be involved in root nodule symbiosis with host plants. The chemistry of the newly described QS signal indicated that there may be a dedicated cellular enzyme involved in its synthesis in addition to the types known for production of other AHLs. Indeed, we report that an additional gene is required for synthesis of the unique signal, and we propose that this is a three-component QS circuit as opposed to the canonical two-component AHL QS circuits. The signaling system is exquisitely selective. The selectivity may be important when this species resides in the complex microbial communities around host plants and may make this system useful in various synthetic biology applications of QS circuits.

RdmA Is a Key Regulator in Autoinduction of DSF Quorum Quenching in Pseudomonas nitroreducens HS-18.

Diffusible signal factor (DSF) represents a family of widely conserved quorum-sensing (QS) signals which regulate virulence factor production and pathogenicity in numerous Gram-negative bacterial pathogens. We recently reported the identification of a highly potent DSF-quenching bacterial isolate, Pseudomonas nitroreducens HS-18, which contains an operon with four DSF-inducible genes, digABCD, or digA-D, that are responsible for degradation of DSF signals. However, the regulatory mechanisms that govern the digA-D response to DSF induction have not yet been characterized. In this study, we identified a novel transcriptional regulator we designated RdmA (regulator of DSF metabolism) which negatively regulates the expression of digA-D and represses DSF degradation. In addition, we found that a gene cluster located adjacent to rdmA was also negatively regulated by RdmA and played a key role in DSF degradation; this cluster was hence named dmg (DSF metabolism genes). An electrophoretic mobility shift assay and genetic analysis showed that RdmA represses the transcriptional expression of the dmg genes in a direct manner. Further studies demonstrated that DSF acts as an antagonist and binds to RdmA, which abrogates RdmA binding to the target promoter and its suppression on transcriptional expression of the dmg genes. Taken together, the results from this study have unveiled a central regulator and a gene cluster associated with the autoinduction of DSF degradation in P. nitroreducens HS-18, and this will aid in the understanding of the genetic basis and regulatory mechanisms that govern the quorum-quenching activity of this potent biocontrol agent. IMPORTANCE DSF family quorum-sensing (QS) signals play important roles in regulation of bacterial physiology and virulence in a wide range of plant and human bacterial pathogens. Quorum quenching (QQ), which acts by either degrading QS signals or blocking QS communication, has proven to be a potent disease control strategy, but QQ mechanisms that target DSF family signals and associated regulatory mechanisms remain largely unknown. Recently, we identified four autoinduced DSF degradation genes (digABCD) in P. nitroreducens HS-18. By using a combination of transcriptome and genetic analysis, we identified a central regulator that plays a key role in autoinduction of dig expression, as well as a new gene cluster (dmgABCDEFGH) involved in DSF degradation. The significance of our study is in unveiling the autoinduction mechanism that governs DSF signal quorum quenching for the first time, to our knowledge, and in identification of new genes and enzymes responsible for DSF degradation. The findings from this study shed new light on our understanding of the DSF metabolism pathway and the regulatory mechanisms that modulate DSF quorum quenching and will provide useful clues for design and development of a new generation of highly potent QQ biocontrol agents against DSF-mediated bacterial infections.

Application of an Autoinduction Strategy to Optimize the Heterologous Production of an Antitumor Bispecific Fusion Protein Based on the TRAIL Receptor-Selective Mutant Variant in Escherichia coli.

Autoinduction is a simple approach for heterologous protein expression that helps to achieve the high-level production of recombinant proteins in soluble form. In this work, we investigated if the application of an autoinduction strategy could help to optimize the production of bifunctional protein SRH-DR5-B, the DR5-specific TRAIL variant DR5-B fused to a VEGFR2-specific peptide SRHTKQRHTALH for dual antitumor and antiangiogenic activity. The protein was expressed in Escherichia coli SHuffle B T7, BL21(DE3), and BL21(DE3)pLysS strains. By IPTG induction, the highest expression level was in SHuffle B T7, while by autoinduction, the similar expression level was achieved in BL21(DE3)pLysS. However, in SHuffle B T7, only 45% of IPTG-induced SRH-DR5-B was expressed in soluble form, in contrast to 75% autoinduced in BL21(DE3)pLysS. The yield of purified SRH-DR5-B protein expressed by autoinduction in BL21(DE3)pLysS was 28 ± 4.5 mg per 200 ml of cell culture, which was 1.4 times higher than the yield from IPTG-induced SHuffle B T7. Regardless of the production method, SRH-DR5-B was equally cytotoxic to BxPC-3 human tumor cells expressing DR5 and VEGFR2 receptors. Thus, the production of SRH-DR5-B by autoinduction in the E. coli BL21(DE3)pLysS strain is an efficient, technologically simple, and economical technique that allows to obtain a large amount of active protein from the cytoplasmic cell fraction. Our work demonstrates that the strategy of induction of protein expression is no less important than the strain selection.

Autoinduction Expression Modules for Regulating Gene Expression in Bacillus subtilis.

Although quorum sensing (QS) promoters that can autonomously activate gene expression have been identified and engineered in Bacillus subtilis, researchers focus on quantifying individual promoters while ignoring the interaction between other genetic regulatory elements. Here, we constructed the autoinduction expression modules consisting of promoters responsive to QS ComQXPA, ribosome binding sites (RBSs), and terminators. Using superfolder green fluorescent protein (sfGFP) as a reporter gene, three individual element libraries were generated from 945 promoters, 12,000 RBSs, and 425 terminators by random mutation, de novo design, and database mining strategies, respectively. Then, the efficiency of three libraries in regulating gene expression was further enhanced by engineering the core region of each optimal element. After hybridizing the element libraries, the generated expression modules exhibited a 627-fold range in regulating gene expression without significantly affecting the autoinduction initiation. Here, the hybrid modules with broad expression strength may benefit the application of QS-based autoinduction systems in B. subtilis.

Analysis of Time-Dependent Pharmacokinetics Using In Vitro-In Vivo Extrapolation and Physiologically Based Pharmacokinetic Modeling.

SCR430, a sorafenib derivative, is an investigational drug exhibiting anti-tumor action. This study aimed to have a mechanistic understanding of SCR430's time-dependent pharmacokinetics (TDPK) through an ex vivo study combined with an in vitro-in vivo extrapolation (IVIVE) and physiologically based pharmacokinetic (PBPK) modeling. A non-compartmental pharmacokinetic analysis was performed after intravenous SCR430 administration in female Sprague-Dawley rats for a control group (no treatment), a vehicle group (vehicle only, 14 days, PO), and a repeated-dosing group (SCR430, 30 mg/kg/day, 14 days, PO). In addition, hepatic uptake and metabolism modulation were investigated using isolated hepatocytes from each group of rats. The minimal PBPK model based on IVIVE was constructed to explain SCR430's TDPK. Repeated SCR430 administration decreased the systemic exposure by 4.4-fold, which was explained by increased hepatic clearance (4.7-fold). The ex vivo study using isolated hepatocytes from each group suggested that the increased hepatic uptake (9.4-fold), not the metabolic activity, contributes to the increased hepatic clearance. The minimal PBPK modeling based on an ex vivo study could explain the decreased plasma levels after the repeated doses. The current study demonstrates the TDPK after repeated dosing by hepatic uptake induction, not hepatic metabolism, as well as the effectiveness of an ex vivo approach combined with IVIVE and PBPK modeling to investigate the TDPK.

Heterogeneity in the spontaneous induction of the promoter of the ColE9 operon in Escherichia coli.

Spontaneous production of E colicins is known to occur in only a small fraction of colicinogenic population. The current study aimed to determine if the same holds true for the production of colicin E9 in real time, by investigating the induction dynamics of the promoter of the ColE9 operon which results in the expression of the ColE9 activity and functional genes. A novel fluorescent reporter was constructed which carries the fusion of the ColE9 promoter and the gfpmut2 gene in a low copy number plasmid that was compatible with the native ColE9-J plasmid. Using the fluorescent reporter construct in the non colicinogenic E. coli cells, the induction of the ColE9 promoter was investigated. The current study demonstrates that the spontaneous induction of the ColE9 promoter occurs in a heterogenous manner and this heterogeneity is maintained in a bacterial population for several generations suggesting that it is unlikely due to any irreversible mutation in the bacterial culture. Furthermore, the same investigations were repeated using the colicin E9 producing E. coli cells. Flow cytometry analysis revealed that 7.1 ± 0.68% of the colicin E9 producing E. coli cells expressed GFP albeit only 2.45 ± 0.30% was observed from non colicinogenic E. coli cells. The considerable increase in the number of the fluorescent cells was likely due to the DNase activity of colicin E9 produced by their clonemates, resulting the auto-induction, which can be abolished with the inactivation of the DNase activity of the colicin E9.

Expression, Purification, and in vitro Enzyme Activity Assay of a Recombinant Aldehyde Dehydrogenase from Thermus thermophilus, Using an Escherichia coli Host.

Based on previous in-depth characterisation, aldehyde dehydrogenases (ALDH) are a diverse superfamily of enzymes, in terms of both structure and function, present in all kingdoms of life. They catalyse the oxidation of an aldehyde to carboxylic acid using the cofactor nicotinamide adenine dinucleotide (phosphate) (NAD(P)+), and are often not substrate-specific, but rather have a broad range of associated biological functions, including detoxification and biosynthesis. We studied the structure of ALDHTt from Thermus thermophilus, as well as performed its biochemical characterisation. This allowed for insight into its potential substrates and biological roles. In this protocol, we describe ALDHTt heterologous expression in E. coli, purification, and activity assay (based on Shortall et al., 2021 ). ALDHTt was first copurified as a contaminant during caa3-type cytochrome oxidase isolation from T. thermophilus. This recombinant production system was employed for structural and biochemical analysis of wild-type and mutants, and proved efficient, yielding approximately 15-20 mg/L ALDHTt. For purification of the thermophilic his-tagged ALDHTt, heat treatment, immobilized metal affinity chromatography (IMAC), and gel filtration chromatography were used. The enzyme activity assay was performed via UV-Vis spectrophotometry, monitoring the production of reduced nicotinamide adenine dinucleotide (NADH). Graphical abstract: Flow chart outlining the steps in ALDHTt expression and purification, highlighting the approximate time required for each step.

Production and Functional Characterization of a Novel Mannanase from Alteromonadaceae Bacterium Bs31.

BACKGROUND: Mannans are the main components of hemicellulose in nature and serve as the major storage polysaccharide in legume seeds. To mine new mannanase genes and identify their functional characteristics are an important basis for mannan biotechnological applications. OBJECTIVE: In this study, a putative mannanase gene (ManBs31) from the genome of the marine bacterium Alteromonadaceae Bs31 was characterized. METHODS: Amino acid sequence analysis and protein structural modeling were used to reveal the molecular features of ManBs31. The catalytic domain of ManBs31 was recombinantly produced using Escherichia coli and Pichia pastoris expression systems. The biochemical properties of the enzymes were determined by reducing sugar assay and thin-layer chromatography. RESULTS: Sequence analysis revealed that ManBs31 was a multidomain protein, consisting of a catalytic domain belonging to glycoside hydrolase family 5 (GH5) and two cellulose-binding domains. Recombinant ManBs31-GH5 exhibited the maximum hydrolytic performance at 70 ºC and pH 6. It showed the best hydrolysis capacity toward konjac glucomannan (specific enzyme activity up to 1070.84 U/mg) and poor hydrolysis ability toward galactomannan with high side-chain modifications (with a specific activity of 344.97 U/mg and 93.84 U/mg to locust bean gum and ivory nut mannan, respectively). The hydrolysis products of ManBs31-GH5 were mannooligosaccharides, and no monosaccharide was generated. Structural analysis suggested that ManBs31-GH5 had a noncanonical +2 subsite compared with other GH5 mannanases. CONCLUSION: ManBs31 was a novel thermophilic endo-mannanase and it provided a new alternative for the biodegradation of mannans, especially for preparation of probiotic mannooligosaccharides.

Simple Scalable Protein Expression and Extraction Using Two-stage Autoinducible Cell Autolysis and DNA/RNA Autohydrolysis in Escherichia coli.

Recombinant protein expression is extensively used in biological research. Despite this, current protein expression and extraction methods are not readily scalable or amenable for high-throughput applications. Optimization of protein expression conditions using traditional methods, reliant on growth-associated induction, is non-trivial. Similarly, protein extraction methods are predominantly restricted to chemical methods, and mechanical methods reliant on expensive specialized equipment more tuned for large-scale applications. In this article, we outline detailed protocols for the use of an engineered autolysis/autohydrolysis E. coli strain, in two-stage fermentations in shake-flasks. This two-stage fermentation protocol does not require optimization of expression conditions and results in high protein titers. Cell lysis in an engineered strain is tightly controlled and only triggered post-culture by addition of a 0.1% detergent solution. Upon cell lysis, a nuclease digests contaminating host oligonucleotides, which facilitates sample handling. This method has been validated for use at different scales, from microtiter plates to instrumented bioreactors. Graphic abstract: Two-stage protein expression, cell autolysis and DNA/RNA autohydrolysis. Reprinted with permission from Menacho-Melgar et al. (2020a). Copyright 2020 John Wiley and Sons.

Simple Extract Preparation Methods for E. coli-Based Cell-Free Expression.

Cell-free protein synthesis (CFPS) is a powerful platform for synthetic biology, allowing for the controlled expression of proteins without reliance on living cells. However, the process of producing the cell extract, a key component of cell-free reactions, can be a bottleneck for new users to adopt CFPS as it requires technical knowledge and significant researcher oversight. Here, we provide a detailed method for implementing a simplified cell extract preparation workflow using CFAI media. We also provide a detailed protocol for the alternative, 2x YPTG media-based preparation process, as it represents a useful benchmark within the cell-free community.

Process Development for the Production and Purification of PEGylated RhG-CSF Expressed in Escherichia coli.

BACKGROUND AND OBJECTIVES: Recombinant human granulocyte-colony stimulating factor (rhG-CSF) and its PEGylated form (PEG-GCSF) are used in cancer therapy. Thus, developing a more cost-effectively method for expressing rhG-CSF and the PEGylation optimization of rhG-CSF by reaction engineering and subsequent purification strategy is necessary. METHODS: RhG-CSF expression in Escherichia coli BL21 (DE3) was carried out by auto-induction batch fermentation and improved for maximizing rhG-CSF productivity. After that, purified rhGCSF was PEGylated using methoxy polyethylene glycol propionaldehydes (mPEG20-ALD). The various conditions effect of extraction and purification of rhG-CSF and PEG-GCSF were assayed. RESULTS: The assessment results revealed that the auto-induction batch cultivation strategy had maximum productivity, and rhG-CSF purity was more than 99%. The obtained data of rhG-CSF PEGylation displayed that the optimized conditions of rhG-CSF PEGylation and purification enhanced homogeneity PEG-GCSF and managed reaction toward optimal yield of PEG-GCSF (70%) and purity of 99.9%. Findings from FTIR, CD, fluorescence spectroscopy, and bioassay revealed that PEGylation was executed exactly in the rhG-CSF N-terminus, and products maintained their conformation properties. CONCLUSION: Overall, the developed approach expanded strategies for high yield rhG-CSF by simplified auto-induction batch fermentation system and rhG-CSF PEGylation, which are simple and timesaving, economical, and high efficiency.

Effect of autoinduction and food on the pharmacokinetics of furmonertinib and its active metabolite characterized by a population pharmacokinetic model.

Furmonertinib (AST2818) is a novel third-generation irreversible EGFR TKI and recently has been approved in China for the treatment of non-small cell lung cancer (NSCLC) with EGFR-sensitizing and T790M resistance mutations. In the current study, we developed a semi-mechanistic population pharmacokinetic model to characterize the nonstationary pharmacokinetics (PK) of the furmonertinib and its active metabolite AST5902 simultaneously. The PK data of furmonertinib and AST5902 were obtained from 38 NSCLC patients and 16 healthy volunteers receiving 20-240 mg furmonertinib in three clinical trials. A nonlinear mixed-effects modeling approach was used to describe the PK data. The absorption process of furmonertinib was described by a transit compartment model. The disposition of both furmonertinib and AST5902 was described by a two-compartment model. An indirect response model accounted for the autoinduction of furmonertinib metabolism mediated by CYP3A4. The model-based simulation suggested that furmonertinib clearance was increased in one cycle of treatment (orally once daily for 21 days) compared to baseline, ranging from 1.1 to 1.8 fold corresponding to the dose range of 20-240 mg. The concentration of furmonertinib was decreased over time whereas that of AST5902 was increased. Interestingly, the concentration of the total active compounds (furmonertinib and AST5902) appeared to be stable. The food intake, serum alkaline phosphatase and body weight were identified as statistically significant covariates. The mechanism of food effect on PK was investigated, where the food intake might increase the bioavailability of furmonertinib via increasing the splanchnic blood flow. Overall, a population PK model was successfully developed to characterize the nonstationary PK of furmonertinib and AST5902 simultaneously. The concentrations of total active compounds were less affected by the autoinduction of furmonertinib metabolism.