Expression Of Fusion Protein Research Articles

In vivo brain delivery of BBB-enabled iduronate 2-sulfatase in rats

BackgroundIduronate-2-sulfatase (IDS) deficiency (MPS II; Hunter syndrome) is a disorder that exhibits peripheral and CNS pathology. The blood brain barrier (BBB) prevents systemic enzyme replacement therapy (ERT) from alleviating CNS pathology. We aimed to enable brain delivery of systemic ERT by using molecular BBB-Trojans targeting endothelial transcytosis receptors. Methods: Single-domain antibody (sdAb)-enzyme fusion protein constructs were prepared in Yarrowia lipolytica. sdAb affinity and BBB permeability were characterized using SPR and an in vitro rodent BBB assay, respectively. In vivo pharmacokinetic (PK) analysis was performed in rats. Quantification of fusion protein amounts were performed using LC-MS.ResultsFusion proteins consisting of IDS and BBB-transmigrating sdAbs, albumin binding sdAbs or human serum albumin (HSA) were evaluated for their in vitro BBB permeability. IGF1R3H5-IDS was selected for in vivo PK analysis in rats. IDS and IGF1R3H5-IDS exhibited very short (< 10 min) serum half-life (t1/2α), while constructs containing either HSA or anti-serum albumin sdAbs (R28 or M79) showed 8–11 fold increases in the area under the curve (AUC) in serum. CSF analysis indicated that IGF1R3H5 increased brain exposure by 9 fold (AUC) and constructs containing HSA or R28 exhibited 42–52 fold increases. Quantitation of brain levels confirmed the increased and sustained delivery of IDS to the brain of HSA- and R28-containing constructs. Lastly, analysis of brain fractions demonstrated that the increases in brain tissue were due to parenchymal delivery without fusion protein accumulation in brain vessels.ConclusionsThese results demonstrate the utility of IGF1R-targeting sdAbs to effect brain delivery of lysosomal enzymes, as well as the utility of serum albumin-targeting sdAbs in t1/2 extension, to increase brain delivery of rapidly cleared enzymes.

Notoginsenoside R1 Attenuates H/R Injury in H9c2 Cells by Maintaining Mitochondrial Homeostasis

Mitochondrial homeostasis is crucial for maintaining cellular energy production and preventing oxidative stress, which is essential for overall cellular function and longevity. Mitochondrial damage and dysfunction often occur concomitantly in myocardial ischemia–reperfusion injury (MIRI). Notoginsenoside R1 (NGR1), a unique saponin from the traditional Chinese medicine Panax notoginseng, has been shown to alleviate MIRI in previous studies, though its precise mechanism remains unclear. This study aimed to elucidate the mechanisms of NGR1 in maintaining mitochondrial homeostasis in hypoxia/reoxygenation (H/R) H9c2 cells. The results showed that NGR1 pretreatment effectively increased cell survival rates post-H/R, reduced lactate dehydrogenase (LDH) leakage, and mitigated cell damage. Further investigation into mitochondria revealed that NGR1 alleviated mitochondrial structural damage, improved mitochondrial membrane permeability transition pore (mPTP) persistence, and prevented mitochondrial membrane potential (Δψm) depolarization. Additionally, NGR1 pretreatment enhanced ATP levels, increased the activity of mitochondrial respiratory chain complexes I–V after H/R, and reduced excessive mitochondrial reactive oxygen species (mitoROS) production, thereby protecting mitochondrial function. Further analysis indicated that NGR1 upregulated the expression of mitochondrial biogenesis-related proteins (PGC-1α, Nrf1, Nrf2) and mitochondrial fusion proteins (Opa1, Mfn1, Mfn2), while downregulating mitochondrial fission proteins (Fis1, Drp1) and reducing mitochondrial autophagy (mitophagy) levels, as well as the expression of mitophagy-related proteins (Pink1, Parkin, BNIP3) post-H/R. Therefore, this study showed that NGR1 can maintain mitochondrial homeostasis by regulating mitophagy, mitochondrial fission–fusion dynamics, and mitochondrial biogenesis, thereby alleviating H9c2 cell H/R injury and protecting cardiomyocytes.

Recombinant Protein Production and Purification of Rift Valley Fever Virus Nucleoprotein from Escherichia coli Expression Systems.

The recombinant expression and purification of viral proteins are a key component in the study of the immune response of viruses, as well as the creation of diagnostic techniques for the detection of viruses. For structurally simple proteins, one commonly used technique is the production of recombinant proteins in bacterial expression systems, which enable the large-scale synthesis and purification of recombinant viral proteins. In this technique, the cDNA encoding for a viral protein is cloned into a bacterial expression vector (with an appropriate purification tag), produced in a modified bacterial culture, and optimized for maximum protein production in a minimal amount of time. In this chapter, a protocol for the production of Rift Valley fever virus nucleoprotein is described. This protein has been previously shown to highly antigenic (and thus, is used in diagnostic tests), and has also been shown to be a potent inducer of the T-cell response following Rift Valley fever virus infection. The protocol outlined in this chapter describes the cloning of the cDNA of RVFV nucleoprotein into a bacterial expression vector, which also contains a fusion protein for optimal protein expression and solubilization, as well as a poly-histidine tag for efficient purification This chapter also describes the steps required for bacterial transformation, culture, lysis, purification and dialysis of RVFV nucleoprotein, resulting in a recombinant protein preparation, which can be upscaled to produce milligram quantities of protein product, which in turn can be used for downstream immunological and diagnostic applications.

Hypoxia Affects Mitochondrial Stress and Facilitates Tumor Metastasis of Colorectal Cancer Through Slug SUMOylation.

Colorectal cancer (CRC) is a malignant tumor. Slug has been found to display a key role in diversified cancers, but its relevant regulatory mechanisms in CRC development are not fully explored. Hence, exploring the function and regulatory mechanisms of Slug is critical for the treatment of CRC. Protein expressions of Slug, N-cadherin, E-cadherin, Snail, HIF-1α, SUMO1, Drp1, Opa1, Mfn1/2, PGC-1α, NRF1, and TFAM were measured through western blot. To evaluate the protein expression of Slug and SUMO-1, an immunofluorescence assay was used. Cell migration ability was tested through transwell assay. The SUMOylation of Slug was examined through CO-IP assay. Slug displayed higher expression and facilitated tumor metastasis in CRC. In addition, hypoxia treatment was discovered to upregulate HIF-1α, Slug, and SUMO-1 levels, as well as induce Slug SUMOylation. Slug SUMOylation markedly affected mitochondrial biosynthesis, fusion, and mitogen-related protein expression levels to trigger mitochondrial stress. Additionally, the induced mitochondrial stress by hypoxia could be rescued by Slug inhibition and TAK-981 treatment. Our study expounded that hypoxia affects mitochondrial stress and facilitates tumor metastasis of CRC through Slug SUMOylation.

Challenges and strategies in the soluble expression of CTA1-(S14P5)4-DD and CTA1-(S21P2)4-DD fusion proteins as candidates for COVID-19 intranasal vaccines.

Developing intranasal vaccines against pandemics and devastating airborne infectious diseases is imperative. The superiority of intranasal vaccines over injectable systemic vaccines is evident, but developing effective intranasal vaccines presents significant challenges. Fusing a protein antigen with the catalytic domain of cholera toxin (CTA1) and the two-domain D of staphylococcal protein A (DD) has significant potential for intranasal vaccines. In this study, we constructed two fusion proteins containing CTA1, tandem repeat linear epitopes of the SARS-CoV-2 spike protein (S14P5 or S21P2), and DD. Structural predictions indicated that each component of the fusion proteins was compatible with its origin. In silico analyses predicted high solubility for both fusion proteins when overexpressed in Escherichia coli. However, contrary to these predictions, the constructs exhibited limited solubility. Lowering the cultivation temperature from 37°C to 18°C did not improve solubility. Inducing expression with IPTG at the early log phase significantly increased soluble CTA1-(S21P2)4-DD but not CTA1-(S14P5)4-DD. Adding non-denaturing detergents (Nonidet P40, Triton X100, or Tween 20) to the extraction buffer significantly enhanced solubility. Despite this, purification experiments yielded low amounts, only 1-2 mg/L of culture, due to substantial losses during the purification stages. These findings highlight the challenges and potential strategies for optimizing soluble expression of CTA1-DD fusion proteins for intranasal vaccines.

The Mitochondrial Fusion Promoter M1 Mitigates Cigarette Smoke-Induced Airway Inflammation and Oxidative Stress via the PI3K-AKT Signaling Pathway.

This study investigated the efficacy and underlying mechanism of the mitochondrial fusion promoter M1 in mitigating cigarette smoking (CS)-induced airway inflammation and oxidative stress both in vitro and in vivo models. Cigarette smoke extract (CSE)-treated airway epithelial cells (BEAS-2B) and CS-exposed mice were pretreated with M1, followed by the measurement of proinflammatory cytokines, oxidative stress, mitochondrial fusion proteins (MFN2 and OPA1) and fission proteins (DRP1 and MFF). Molecular pathways were elucidated through transcriptomic analysis and Western blotting. M1 pretreatment in CSE-treated cells significantly reduced the release of inflammatory cytokines (interleukin (IL)-6, IL-8 and tumor necrosis factor (TNF)-α); reduced malondialdehyde (MDA) and reactive oxygen species (ROS) levels; increased superoxide dismutase (SOD) activity; protected mitochondrial function by increasing the expression of mitochondrial fusion proteins (MFN2 and OPA1) and decreasing the expression of mitochondrial fission proteins (DRP1 and MFF). M1 attenuated CS-induced lung histologic damage and mucus hypersecretion in mice, relieved high oxidative stress and reduced the release of IL-6 and IL-8 in BALF. Similarly, it also protected mitochondrial function by regulating the CS-induced imbalance of mitochondrial dynamic proteins. Transcriptome sequencing and Western blotting showed that M1 inhibited CSE- or CS-induced activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT) signaling pathway. M1 plays a protective role in inflammation, oxidative stress and mitochondrial dynamics dysfunction caused by CS by inhibiting the PI3K-AKT signaling pathway; thus, it has therapeutic potential for the treatment of CS-induced airway disorders.

Heterologous Expression and Polyphasic Analysis of CLA-Converting Linoleic Acid Isomerase from Bifidobacterium breve JKL2022.

The probiotic Bifidobacterium breve is known for its efficient conjugated linoleic acid (CLA) conversion, yet their CLA conversion pathway remains underexplored. This study investigated B. breve JKL2022 for its CLA conversion in actively growing cells, washed cell states, and in crude protein extracts. Moreover, the study aimed to confirm the CLA-converting enzyme in strain JKL2022 and optimize its purification through heterologous expression of fusion proteins (LAI_sGFP and MBP_LAI). JKL2022 exhibited superior CLA conversion compared to genetically similar B. breve strains (JCM7017, JCM7019, JCM1192, and JCM1273), particularly the observed CLA conversion in washed cells (60.14 ± 7.60%) and crude protein fractions (96.11 ± 6.63%). The multipass transmembrane linoleic acid isomerase (LAI) was cloned into the E. coli BL21(DE3) as free LAI or modified with superfolder-GFP or MBP tags and expressed with 0.01 mM IPTG at 37 °C, resulting in highly active protein fractions. LAI was characterized by predictive modeling, molecular docking, and phylogenetic analyses. Moreover, reverse transcription-quantitative PCR analysis revealed upregulation (20-140× higher expression) of lai in JKL2022 compared with that in the JCM strains. Nevertheless, upscaling the production and purification of LAI for downstream applications remains a challenge, primarily because of their membrane-spanning configuration.

Identification of linear B cell epitopes on the E146L protein of African swine fever virus with monoclonal antibodies

The outbreak and spread of African swine fever virus (ASFV) have caused considerable economic losses to the pig industry worldwide. Currently, to promote the development of effective ASF vaccines, especially subunit vaccines, more antigenic protein targets are urgently needed. In this work, six transmembrane proteins (I329L, E146L, C257L, EP153R, I177L, and F165R) were expressed in mammalian cell lines and screened with pig anti-ASFV serum. It was found that the E146L protein was an immunodominant protein antigen among the six selected proteins. Moreover, the E146L protein induced antibody responses in all immunized pigs. To gain insight into the antigenic characteristics of the E146L protein, three monoclonal antibodies (mAbs; 12H12, 15G1, and 15H10) were generated by immunizing BALB/c mice with the purified E146L protein. The epitopes of the mAbs were further finely mapped through a peptide fusion protein expression strategy. Finally, the epitopes of the mAbs were identified as 48PDESSIAYMRFRN61 of the mAb 12H12, 138TLTGLQRII146 of the mAb 15G1, and 30GWSPFKYSKGNT41 of the mAb 15H10. Furthermore, the epitope of mAb 15H10 was validated as the immunodominant epitope with ASFV-infected pig sera. The chemically synthesized mAb 15H10 epitope peptide (EP1) exhibited the most extensive immunoreactivity with artificially or naturally ASFV-infected pig sera. The epitope 15H10 is located on the surface of the E146L protein and is highly conserved. These findings provide insight into the structure and function of the E146L protein of ASFV.

Expression, purification and characterization of a novel triple fusion protein developed for the immunotherapy of survivin positive cancers

Survivin is an inhibitor of apoptosis, and expressed in a large number of cancers. As Survivin expression is very low in normal tissues, it assumes significance as a prominent target for tumor diagnosis, prognosis and developing anti-cancer therapies. We report development of a novel triple fusion protein for a prospective vaccine against Survivin in targeted cancer immunotherapy. A gene was synthesized by combining the nucleotides encoding human origin Survivin and heat-labile enterotoxin of Escherichia coli (LTB). Further, nucleotides corresponding to single chain variable fragment (scFv) of a monoclonal having affinity for DEC205 receptor present on dendritic cells, were also incorporated into the gene sequence. This complete gene was expressed to a triple fusion recombinant protein using a bacterial expression vector under the control of robust bacteriophage T7 promoter. The recombinant DCSurvivin-LTB protein, with a size of approximately 60 kDa, was purified from the inclusion bodies using affinity based Ni-NTA columns. The purified protein was confirmed by the Western blot, and further characterized with circular dichroism, fluorescence spectroscopy and mass spectroscopy. This molecularly adjuvanted Survivin fusion protein designed to deliver to the dendritic cells for better antigen processing, elicited a stronger anti-Survivin immune response compared to Survivin protein alone. It can be an effective vaccine in active and passive immunotherapies for Survivin expressing cancer cells.

In vivo thrombin activity in the diatom Phaeodactylum tricornutum: biotechnological insights.

Diatoms are responsible for 20% of global carbon dioxide fixation and have significant potential in various biotechnological and industrial applications. Recently, the pennate diatom Phaeodactylum tricornutum has emerged as a prominent platform organism for metabolic engineering and synthetic biology. The availability of its genome sequence has facilitated the development of new bioengineering tools. In this study, we used in silico analyses to identify sequences potentially encoding thrombin-like proteins, which are involved in recognizing and cleaving the thrombin sequence LVPRGS in P. tricornutum. Protein structure prediction and docking studies indicated a similar active site and ligand positioning compared to characterized human and bovine thrombin. The evidence and efficiency of the cleavage were determined in vivo using two fusion-protein constructs that included YFP to measure expression, protein accumulation, and cleavage. Western blot analysis revealed 50-100% cleavage between YFP and N-terminal fusion proteins. Our findings suggest the existence of a novel thrombin-like protease in P. tricornutum. This study advances the application of diatoms for the synthesis and production of complex proteins and enhances our understanding of the functional role of these putative thrombin sequences in diatom physiology. KEY POINTS: • Protein structure predictions reveal thrombin-like active sites in P. tricornutum. • Validated cleavage efficiency of thrombin-like protease on fusion proteins in vivo. • Study advances bioengineering tools for diatom-based biotechnological applications.

PHD-BAH Domain in ASH1L Could Recognize H3K4 Methylation and Regulate the Malignant Behavior of Cholangiocarcinoma.

Histone methyltransferase absent, small, or homeotic discs1-like (ASH1L) is composed of su(var)3-9, enhancer of zeste, trithorax (SET) domain, pleckstrin homology domain (PHD) domain, middle (MID) domain, and bromo adjacent homology (BAH) domain. The SET domain of ASH1L is known to mediate mediate H3K36 dimethylation (H3K36me2) modification. However, the specific functions of the PHD-BAH domain remain largely unexplored. This study aimed to explore the biological function of the PHD-BAH domain in ASH1L. We employed a range of techniques, including a prokaryotic fusion protein expression purification system, pull-down assay, Isothermal Titration Calorimetry (ITC), polymerase chain reaction (PCR), and sitedirected mutagenesis, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas9) gene editing, cell culture experiment, western blot, cell proliferation assay, and cell apoptosis test. The PHD-BAH domain in ASH1L preferentially binds to the H3K4me2 peptide over H3K4 monomethylation (H3K4me1) and H3K4 trimethylation (H3K4me3) peptide. Notably, the W2603A mutation within the PHD-BAH domain could disrupt the interaction with H3K4me2 in vitro. Compared with wild-type Cholangiocarcinoma (CHOL) cells, deletion of the PHD-BAH domain in ASH1L led to increased CHOL cell apoptosis and reduced cell proliferation (P < 0.001). Additionally, the W2603A mutation affected the regulation of the proteasome 20S subunit beta (PSMB) family gene set. W2603A mutation was crucial for the interaction between the PHD-BAH domain and the H3K4me2 peptide. ASH1L regulated CHOL cell survival and proliferation through its PHD-BAH domain by modulating the expression of the PSMB family gene set.

Apigenin Ameliorates H2O2-Induced Oxidative Damage in Melanocytes through Nuclear Factor-E2-Related Factor 2 (Nrf2) and Phosphatidylinositol 3-Kinase (PI3K)/Protein Kinase B (Akt)/Mammalian Target of Rapamycin (mTOR) Pathways and Reducing the Generation of Reactive Oxygen Species (ROS) in Zebrafish

Background: Apigenin is one of the natural flavonoids found mainly in natural plants, as well as some fruits and vegetables, with celery in particular being the most abundant. Apigenin has antioxidant, anti-tumor, anti-inflammatory, and anticancer effects. In this research, we attempted to further investigate the effects of apigenin on the mechanism of repairing oxidative cell damage. The present study hopes to provide a potential candidate for abnormal skin pigmentation disorders. Methods: We used 0.4 mM H2O2 to treat B16F10 cells for 12 h to establish a model of oxidative stress in melanocytes, and then we gave apigenin (0.1~5 μM) to B16F10 cells for 48 h, and detected the expression levels of melanin synthesis-related proteins, dendritic regulation-related proteins, antioxidant signaling pathway- and Nrf2 signaling pathway-related proteins, autophagy, and autophagy-regulated pathways by immunoblotting using Western blotting. The expression levels of PI3K/Akt/mTOR proteins were measured by β-galactosidase staining and Western blotting for cellular decay, JC-1 staining for mitochondrial membrane potential, and Western blotting for mitochondrial fusion- and mitochondrial autophagy-related proteins. Results: Apigenin exerts antioxidant effects by activating the Nrf2 pathway, and apigenin up-regulates the expression of melanin synthesis-related proteins Tyr, TRP1, TRP2, and gp100, which are reduced in melanocytes under oxidative stress. By inhibiting the expression of senescence-related proteins p53 and p21, and delaying cellular senescence, we detected the mitochondrial membrane potential using JC-1, and found that apigenin improved the reduction in mitochondrial membrane potential in melanocytes under oxidative stress, and maintained the normal function of mitochondria. In addition, we further detected the key regulatory proteins of mitochondrial fusion and division, MFF, p-DRP1 (S637), and p-DRP1 (S616), and found that apigenin inhibited the down-regulation of fusion-associated protein, p-DRP1 (S637), and the up-regulation of division-associated proteins, MFF and p-DRP1 (S616), due to oxidative stress in melanocytes, and promoted the mitochondrial fusion and ameliorated the imbalance between mitochondrial division and fusion. We further detected the expression of fusion-related proteins OPA1 and Mitofusion-1, and found that apigenin restored the expression of the above fusion proteins under oxidative stress, which further indicated that apigenin promoted mitochondrial fusion, improved the imbalance between mitochondrial division and fusion, and delayed the loss of mitochondrial membrane potential. Apigenin promotes the expression of melanocyte autophagy-related proteins and the key mitochondrial autophagy proteins BNIP3L/Nix under oxidative stress, and activates the PINK1/Parkin signaling pathway by up-regulating the expression of autophagy-related proteins, as well as the expression of PINK1 and Parkin proteins, to promote melanocyte autophagy and mitochondrial autophagy. Conclusions: Apigenin exerts anti-melanocyte premature aging and detachment effects by promoting melanin synthesis, autophagy, and mitochondrial autophagy in melanocytes, and inhibiting oxidative cell damage and senescence.

De novo biosynthesis of β-Arbutin in Komagataella phaffii based on metabolic engineering strategies

Backgroundβ-Arbutin, found in the leaves of bearberry, stands out as one of the globally acknowledged eco-friendly whitening additives in recent years. However, the natural abundance of β-Arbutin is low, and the cost-effectiveness of using chemical synthesis or plant extraction methods is low, which cannot meet the requirements. While modifying the β-Arbutin synthesis pathway of existing strains is a viable option, it is hindered by the limited synthesis capacity of these strains, which hinders further development and application.ResultsIn this study, we established a biosynthetic pathway in Komagataella phaffii for β-Arbutin production with a titer of 1.58 g/L. Through diverse metabolic strategies, including fusion protein construction, enhancing shikimate pathway flux, and augmenting precursor supplies (PEP, E4P, and UDPG), we significantly increased β-Arbutin titer to 4.32 g/L. Further optimization of methanol concentration in shake flasks led to a titer of 6.32 g/L titer after 120 h of fermentation, representing a fourfold increase over the initial titer. In fed-batch fermentation, strain UA3-10 set a record with the highest production to date, reaching 128.6 g/L in a 5 L fermenter.ConclusionsThis is the highest yield in the fermentation tank level of using microbial cell factories for de novo synthesis of β-Arbutin. Applying combinatorial engineering strategies has significantly improved the β-Arbutin yield in K. phaffii and is a promising approach for synthesizing functional products using a microbial cell factory. This study not only advances low-cost fermentation-based production of β-Arbutin but also establishes K. phaffii as a promising chassis cell for synthesizing other aromatic amino acid metabolites.Graphical abstract

Molecular farming expression of recombinant fusion proteins applied to skincare strategies.

This review discusses the current research progress in molecular farming technology in the field of skincare, with an emphasis on molecular farming expression strategies. The strategies of transdermal drug delivery and their advantages are also highlighted. The expression of cosmetically relevant fused proteins has become an important way to enhance the efficacy of the proteins. Therefore, we also discuss the feasibility and strategies for expressing fusion proteins in A. thaliana, specifically the fusion of Epidermal growth factor (EGF) to a cell-penetrating peptide (CPP), in which the production can be greatly enhanced via plant expression systems since these systems offer higher biosecurity, flexibility, and expansibility than prokaryotic, animal and mammalian expression systems. While the fusion of EGF to CCP can enhance its transdermal ability, the effects of the fusion protein on skin repair, melasma, whitening, and anti-aging are poorly explored. Beyond this, fusing proteins with transdermal peptides presents multiple possibilities for the development of tissue repair and regeneration therapeutics, as well as cosmetics and beauty products. As certain plant extracts are known to contain proteins beneficial for skin health, the expression of these proteins in plant systems will better maintain their integrity and biological activities, thereby facilitating the development of more effective skincare products.

Production of recombinant human insulin from a promising Pseudomonas fluorescens cell factory and its kinetic modeling

Insulin intake is recommended for diabetics in addition to a proper diet and lifestyle to maintain adequate blood glucose level. Currently, there is a need for an alternative expression system for insulin production as the current expression systems may not meet the growing demand due to various constraints. Here, we demonstrate the synthesis of human insulin in an unconventional expression system based on Pseudomonas fluorescens, a BSL 1 bacterium. Human insulin was produced in the form of proinsulin fused with fusion protein. Then, the proinsulin fusion protein was purified using Ni-NTA chromatography and converted into human insulin. The physicochemical parameters for producing proinsulin fusion protein are optimized. Glucose and ammonium chloride are determined to be suitable carbon and nitrogen sources, respectively. The validity of insulin and proinsulin fusion protein is assessed using western blot and quantified using ELISA techniques. Up to 145.35 mg/l of the proinsulin fusion protein is achieved at the shake flask level. Further, MALDI-TOF and RP-HPLC analysis of the purified human insulin were observed to be close to the theoretical value and insulin standard, respectively. The expression of the recombinant fusion protein was found to be 214.7 mg/l in a batch bioreactor, a ∼48% enhancement over the shake flask level. Further, kinetic modeling was performed to understand the system regarding growth, substrate utilization and product formation, and to estimate the various kinetic parameters. This study establishes the potential of the P. fluorescens expression system for producing human insulin.

Critical residues in the Ku70 von Willebrand A domain mediate Ku interaction with the LigIV-XRCC4 complex in non-homologous end-joining

The Ku heterodimer (Ku70/Ku80) is central to the non-homologous end-joining (NHEJ) pathway. Ku binds to the broken DNA ends and promotes the assembly of the DNA repair complex. The N-terminal Ku70 von Willebrand A (vWA) domain is known to mediate protein-protein interactions important for the repair process. In particular, the D192 and D195 residues within helix 5 of the Ku70 vWA domain were shown to be essential for NHEJ function, although the precise role of these residues was not identified. Here, we set up a miniTurbo screening system to identify Ku70 D192/D195 residue-specific interactors in a conditional, human Ku70-knockout cell line in response to DNA damage. Using fusion protein constructs of Ku70 wild-type and mutant (D192A/D195R) with miniTurbo, we identified a number of candidate proximal interactors in response to DNA damage treatment, including DNA Ligase IV (LigIV), a known and essential NHEJ complex member. Interestingly, LigIV was enriched in our wildtype screen but not the Ku70 D192A/D195R screen, suggesting its interaction is disrupted by the mutation. Validation experiments demonstrated that the DNA damage-induced interaction between Ku70 and LigIV was disrupted by the Ku70 D192A/D195R mutations. Our findings provide greater detail about the interaction surface between the Ku70 vWA domain and LigIV and offer strong evidence that the D192 and D195 residues are important for NHEJ completion through an interaction with LigIV. Altogether, this work reveals novel potential proximal interactors of Ku in response to DNA damage and identifies Ku70 D192/D195 residues as essential for LigIV interaction with Ku during NHEJ.

Quantitative Analysis of Rhodobacter sphaeroides Storage Organelles via Cryo-Electron Tomography and Light Microscopy.

Bacterial cytoplasmic organelles are diverse and serve many varied purposes. Here, we employed Rhodobacter sphaeroides to investigate the accumulation of carbon and inorganic phosphate in the storage organelles, polyhydroxybutyrate (PHB) and polyphosphate (PP), respectively. Using cryo-electron tomography (cryo-ET), these organelles were observed to increase in size and abundance when growth was arrested by chloramphenicol treatment. The accumulation of PHB and PP was quantified from three-dimensional (3D) segmentations in cryo-tomograms and the analysis of these 3D models. The quantification of PHB using both segmentation analysis and liquid chromatography and mass spectrometry (LCMS) each demonstrated an over 10- to 20-fold accumulation of PHB. The cytoplasmic location of PHB in cells was assessed with fluorescence light microscopy using a PhaP-mNeonGreen fusion-protein construct. The subcellular location and enumeration of these organelles were correlated by comparing the cryo-ET and fluorescence microscopy data. A potential link between PHB and PP localization and possible explanations for co-localization are discussed. Finally, the study of PHB and PP granules, and their accumulation, is discussed in the context of advancing fundamental knowledge about bacterial stress response, the study of renewable sources of bioplastics, and highly energetic compounds.

Foam fractionation studies of recombinant human apolipoprotein A-I

Apolipoprotein A-I (apoA-I), the primary protein component of plasma high-density lipoproteins (HDL), is comprised of two structural regions, an N-terminal amphipathic α-helix bundle domain (residues 1–184) and a hydrophobic C-terminal domain (residues 185–243). When a recombinant fusion protein construct [bacterial pelB leader sequence – human apoA-I (1–243)] was expressed in Escherichia coli shaker flask cultures, apoA-I was recovered in the cell lysate. By contrast, when the C-terminal domain was deleted from the construct, large amounts of the truncated protein, apoA-I (1–184), were recovered in the culture medium. Consequently, following pelB leader sequence cleavage in the E. coli periplasmic space, apoA-I (1–184) was secreted from the bacteria. When the pelB-apoA-I (1–184) fusion construct was expressed in a 5 L bioreactor, substantial foam production (~30 L) occurred. Upon foam collection and collapse into a liquid foamate, SDS-PAGE revealed that apoA-I (1–184) was the sole major protein present. Incubation of apoA-I (1–184) with phospholipid vesicles yielded reconstituted HDL (rHDL) particles that were similar in size and cholesterol efflux capacity to those generated with full-length apoA-I. Mass spectrometry analysis confirmed that pelB leader sequence cleavage occurred and that foam fractionation did not result in unwanted protein modifications. The facile nature and scalability of bioreactor-based apolipoprotein foam fractionation provide a novel means to generate a versatile rHDL scaffold protein.

Computational modeling study of IL-15-NGR peptide fusion protein: a targeted therapeutics for hepatocellular carcinoma

The primary challenge to improving existing cancer treatment is to develop drugs that specifically target tumor cell. NGR peptide is tumor homing peptide that selectively target cancer cells while interleukin 15 is a pleiotropic cytokine with anticancer properties. This study computationally engineered a IL15-NGR fusion peptide by linking the homing peptide NGR with the targeting peptide IL-15. After evaluating and validating the chimeric peptide, we docked it to the IL-15Rα/IL-15Rβ/γc heterodimer receptor, examining the interactions and binding energy and lastly, molecular dynamics simulations were performed. The secondary and tertiary structures, along with physicochemical properties of the designed IL-15-NGR chimeric protein, were predicted using GOR IV, trRosetta and ProtParam online servers respectively. The quality and 3D structure validation were confirmed via ProSA-web and SAVES 6.0 analysis which predicted an ERRAT score of 96.72%, with 97.6% of residues in the Ramachandran plot, validating its structure. Finally, Docking, MD simulations and interaction analysis were performed using ClusPro 2.0 and GROMACS and PDBsum, which exhibited significant hydrogen bonding and salt bridges, confirming the formation of a stable docked complex. These results were further corroborated by simulation analysis, which demonstrated a stable and dynamic behavior of the docked complex in a biological environment. The predicted high expression value of fusion protein was 0.844 in E.coli using SOLUPROT tool. These findings suggest efficient expression of the IL15-NGR fusion protein if its gene is inserted into E. coli and indicates its potential as a safe and effective anticancer treatment, paving the way for targeted therapeutic interventions.Graphical abstract

Induction of DNA single- and double-strand breaks by excited intra- or extracellular green fluorescent protein

Green fluorescent protein (GFP) has opened vast new avenues in studies of live cells and is generally perceived as a benign, nontoxic and harmless fluorescent tag. We demonstrat that excited GFP is capable of inducing substantial DNA damage in cells expressing fusion proteins. In the presence of GFP, even low doses of blue light (12 μJ) induce single strand breaks (SSBs). When the fluorescence of GFP located in the cell nucleus or in the cytoplasm is excited by a much higher dose (17 mJ), DNA double-strand breaks (DSBs) are also induced. Such breaks are induced even when GFP is placed and illuminated in culture medium outside of living cells. We demonstrate that DNA damage is induced by singlet oxygen, which is generated by excited GFP. Although short exposures of live cells to exciting light typically used in fluorescence microscopy induce SSBs but carry little risk of inducing DNA double-strand breaks, larger doses, which may be used in FRAP, FLIM, FCS and super-resolution fluorescence microscopy studies, are capable of inducing not only numerous SSBs but also DSBs.