Aldo-keto reductase family 1 member A1 (AKR1A1) exerts a protective function in alcohol-associated liver disease by reducing 4-HNE accumulation and p53 activation.

BACKGROUND: The development of alcohol-associated liver disease (ALD) is influenced by the amount and duration of alcohol consumption. The resulting liver damage can range from reversible stages, such as steatosis, steatohepatitis and alcoholic fibrosis, to the advanced and irreversible stage of cirrhosis. Aldo-keto reductase family 1 member A1 (AKR1A1) is a member of the aldo-keto reductase family that catalyzes the reduction of aldehyde groups to their corresponding alcohols in an NADPH-dependent manner. AKR1A1 was found to be downregulated in patients diagnosed with ALD. This study aims to interpret the protective effects of AKR1A1 on the development of ALD. METHODS: A 5% alcohol-fed (AF) Akr1a1 knockout (Akr1a1-/-) mouse model and an AML12 hepatocyte model were used. The effects of AKR1A1 on liver function, inflammation, oxidative stress, lipid accumulation, and fibrosis were assessed by ELISA, western blotting, RT‒PCR, and a variety of histological staining methods in AF-induced wild-type (WT) and Akr1a1-/- mice compared to control liquid diet-fed (PF) WT and Akr1a1-/- mice. RESULTS: The results demonstrated that AF-WT mice expressed higher levels of AKR1A1 than WT mice fed a control diet, and they did not show any noticeable liver steatosis. However, AF-Akr1a1-/- mice displayed a lower survival rate and more severe liver injury than AF-WT mice, as demonstrated by increased proinflammatory cytokines, oxidative stress, lipid accumulation, fibrosis, and reduced antioxidant enzymes in their livers. Additionally, elevated levels of 4-HNE and p53 phosphorylation were observed in AF-Akr1a1-/- mice, suggesting that the loss of AKR1A1 led to increased 4-HNE accumulation and subsequent activation of p53, which contributed to the progression of ALD. Furthermore, in AML12 hepatocytes, Akr1a1 knockdown aggravated oxidative stress and steatosis induced by palmitic acid/oleic acid (P/O) inflammation induced by lipopolysaccharide (LPS), and fibrosis induced by TGF-ß1. CONCLUSIONS: This loss-of-function study suggests that AKR1A1 plays a liver-protective role during chronic alcohol consumption by reducing the accumulation of 4-HNE and inhibiting 4-HNE-mediated p53 activation.

Kefir peptides attenuate atherosclerotic vascular calcification and osteoporosis in atherogenic diet-fed ApoE -/- knockout mice.

Aims: Vascular calcification (VC) and osteoporosis were previously considered two distinct diseases. However, current understanding indicates that they share common pathogenetic mechanisms. The available medicines for treating VC and osteoporosis are limited. We previously demonstrated that kefir peptides (KPs) alleviated atherosclerosis in high-fat diet (HFD)-induced apolipoprotein E knockout (ApoE -/- ) mice. The present study further addressed the preventive effects of KPs on VC and osteoporosis in ApoE -/- mice fed a high-cholesterol atherogenic diet (AD). Main methods: Seven-week-old ApoE -/- and wild-type C57BL/6 mice were randomly divided into five groups (n = 6). The development of VC and osteoporosis was evaluated after AD feeding for 13 weeks in KP-treated ApoE -/- mice and compared to C57BL/6 and ApoE -/- mice fed a standard chow diet (CD). Key findings: The results indicated that KP-treated ApoE -/- mice exhibited lower serum total cholesterol, oxidized low-density lipoprotein (ox-LDL), malondialdehyde (MDA) levels, and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatine kinase (CK) activities, which suggested that KPs prevented hyperlipidemia and possible damages to the liver and muscle in ApoE -/- mice. KPs reduced serum tumor necrosis factor-α (TNF-α) and the local expression of TNF-α, IL-1ß, and macrophage-specific CD68 markers in aortic tissues, which suggested that KPs inhibited inflammatory responses in AD-fed ApoE -/- mice. KPs reduced the deposition of lipid, collagen, and calcium minerals in the aortic roots of AD-fed ApoE -/- mice, which suggested that KPs inhibited the calcific progression of atherosclerotic plaques. KPs exerted osteoprotective effects in AD-fed ApoE -/- mice, which was evidenced by lower levels of the bone resorption marker CTX-1 and higher levels of the bone formation marker P1NP. KPs improved cortical bone mineral density and bone volume and reduced trabecular bone loss in femurs. Significance: The present data suggested that KPs attenuated VC and osteoporosis by reducing oxidative stress and inflammatory responses in AD-fed ApoE -/- mice. Our findings contribute to the application of KPs as preventive medicines for the treatment of hyperlipidemia-induced vascular and bone degeneration.

Kefir peptides promote osteogenic differentiation to enhance bone fracture healing in rats.

AIMS: Fractures are the result of fragile bone structures after trauma caused by direct or indirect external impact or strong muscular contraction. Most fracture patients undergo surgical fixation to accelerate the healing process and restore the function of mutilated bone. Promoting the healing process remains an important issue for the treatment of bone fractures. Our previous studies demonstrated the remarkable bone-protective effects of kefir peptides (KPs) in ovariectomized rats and mice. In this study, we further evaluate the efficacy of KPs on fracture healing using a rat model of femoral fracture. MAIN METHODS: Fifteen 8-week-old male Sprague Dawley (SD) rats were divided into the sham, mock, and KPs groups, in which the mock and the KPs groups underwent femur-fracture surgery with nail fixation, while the sham group underwent a sham operation. The next day, rats were orally administered with daily 400 mg/kg of KPs (KPs group) or distilled water (sham and mock groups) for four weeks. X-ray imaging, histochemical staining and serum osteogenic markers were applied for fracture healing evaluation. In vitro, mouse bone marrow mesenchymal stem cells (BMMSCs) and MC3T3-E1 line were subjected to osteoblast differentiation in the presence of KPs and compared with no KPs treatment. KEY FINDINGS: The results demonstrated that KPs treatment improved the progression of the fracture healing process (p < 0.05) and significantly increased the expressions of Col1a1, Alp, Spp1, Vegfa and Cox2 mRNA in the femurs of the KPs-treated fractured rats compared to those of the mock-treated fracture rats. In vitro, KPs treatment promoted bone regeneration factor (Col1a1, Alp, M-csf and Phospho1) expression in MC3T3-E1-derived osteoblast cultures (on Day 3) and enhanced osteogenic differentiation and mineralization in BMMSC-derived osteoblast cultures (on Day 17 and Day 21). SIGNIFICANCE: This is the first study to show that KPs can help with fracture healing by promoting osteogenic differentiation, and it also suggests that KPs can be used as a nutritional supplement to accelerate fracture healing.

Kefir peptides ameliorate osteoporosis in AKR1A1 knockout mice with vitamin C deficiency by promoting osteoblastogenesis and inhibiting osteoclastogenesis.

The AKR1A1 protein is a member of the aldo-keto reductase superfamily that catalyzes the transformation of D-glucuronate to L-gulonate in the synthesis of L-ascorbic acid (vitamin C, Vit C). We previously demonstrated that AKR1A1 knockout mice (AKR1A1eGFP/eGFP) with Vit C deficiency exhibited aberrant bone formation and osteoporosis. In this study, we aimed to evaluate the osteoprotective effects of kefir peptides (KPs) in AKR1A1eGFP/eGFP mice and uncover the underlying mechanism of KPs in the modulation of bone remodeling. Six male CD-1 mice and 24 male AKR1A1eGFP/eGFP mice were used in this study, in which the AKR1A1eGFP/eGFP mice were randomly divided into four groups (n = 6). KPs treatment for 12 weeks exerted several effects in AKR1A1eGFP/eGFP mice including the reduction of serum proinflammatory cytokines (IL-1ß, IL-6, TNF-α), bone resorption markers (CTX-1, RANKL), and the increase of serum bone formation markers (P1NP, OPG, OC). µ-CT analysis indicated that KPs prevented the bone loss in the femurs of AKR1A1eGFP/eGFP mice by significantly increasing the trabecular parameters of bone mineral density, bone volume and bone number. Nanoindentation analysis demonstrated that KPs enhanced the elasticity and hardness of femoral cortical bones in AKR1A1eGFP/eGFP mice. KPs promoted bone marrow mesenchymal stem cells (BMMSCs)-derived osteoblast differentiation and mineralization by upregulating positive regulators of osteoblastogenesis (Runx2, ß-catenin, BMP-2, NFATc1). Conversely, KPs inhibited bone marrow macrophages (BMMs)-derived osteoclast differentiation and bone resorption, which was demonstrated by the facts that KPs suppressed RANKL-induced p38, NF-κB, Akt, PLCγ2 and CREB-1 phosphorylation, decreased the nuclear translocation of NFATc1 and c-Fos. Our findings demonstrate the efficacy of KPs in the prevention of osteoporosis in AKR1A1eGFP/eGFP mice and also unveil the dual effects of KPs in osteogenic promotion and osteoclastic inhibition. This study supports the use of KPs as nutritional supplements for the prevention of osteoporosis.

Therapeutic Effects of Kefir Peptides on Hemophilia-Induced Osteoporosis in Mice With Deficient Coagulation Factor VIII.

Osteoporosis is a clinically prevalent comorbidity in patients with hemophilia. A preventive effect of kefir peptides (KPs) on postmenopausal osteoporosis has been proved. The aim of this study was to evaluate the therapeutic effect of KPs for the treatment of osteoporosis in coagulation factor VIII (FVIII) gene knockout mice (F8KO), a model of hemophilia A. In this study, male F8KO mice at 20 weeks of age were orally administered different doses of KPs for 8 weeks. The therapeutic effects of KPs were shown in the femoral trabeculae and the 4th lumbar vertebrae, which increased the trabecular bone mineral density (BMD), bone volume (Tb.BV/TV), and trabecular number (Tb.N) and decreased the trabecular separation (Tb.Sp), and they were also observed in the femoral cortical bones, in which the mechanical properties were enhanced in a dose-dependent manner. Characterization of receptor activator of nuclear factor κB ligand (RANKL), osteoprotegerin (OPG), and interleukin 6 (IL-6) demonstrated that the serum RANKL/OPG ratio and IL-6 levels were significantly decreased in the F8KO mice after the KP treatment. Tartrate-resistant acid phosphatase (TRAP) staining of mature osteoclasts indicated that the therapeutic effect of KPs in F8KO mice was associated with the functions of KPs to inhibit RANKL-induced osteoclastogenesis by reducing serum RANKL/OPG ratio and IL-6 secretion. The present study is the first to address the potentials of KPs for the treatment of hemophilia-induced osteoporosis in mice and it also provides useful information for the application of KPs as a complementary therapy for the treatment of osteoporosis in hemophilic patients.

KFP-1, a Novel Calcium-Binding Peptide Isolated from Kefir, Promotes Calcium Influx Through TRPV6 Channels.

INTRODUCTION: Kefir is an acidic and alcoholic fermented milk product with multiple health-promoting benefits. A previous study demonstrated that kefir enhanced calcium absorption in intestinal Caco-2 cells. In this study, kefir-fermented peptide-1 (KFP-1) is isolated from the kefir peptide fraction, and its function as a calcium-binding peptide is characterized. METHODS AND RESULTS: KFP-1 was identified as a 17-residue peptide with a sequence identical to that of κ-casein (residues 138-154) in milk protein. KFP-1 is demonstrated to promote calcium influx in Caco-2 and IEC-6 small intestinal cells in a concentration-dependent manner. TRPV6, but not L-type voltage-gated calcium channels, is associated with the calcium influx induced by KFP-1. An in vitro calcium binding assay indicates that the full-length KFP-1 peptide has a higher calcium-binding capacity than the two truncated KFP-1 peptides, KFP-1∆C5 and KFP-1C5. Alexa Fluor 594 labeling shows that KFP-1 is taken up by Caco-2 cells and interacts with calcium ions and TRPV6 protein. Moreover, KFP-1 is found moderately resistant to pepsin and pancreatin digestions and enhanced calcium uptake by intestinal enterocytes in vivo. CONCLUSION: These data suggest that KFP-1, a novel calcium-binding peptide, binds extracellular calcium ions and enters Caco-2 and IEC-6 cells, and promotes calcium uptake through TRPV6 calcium channels. The present study is of great importance for developing kefir-derived metal ion-binding peptides as functional nutraceutical additives.

Benefits of Metformin Combined with Pemetrexed-Based Platinum Doublets as a First-Line Therapy for Advanced Lung Adenocarcinoma Patients with Diabetes.

Lung cancer remains a challenge in daily practice. Chemotherapy is first considered for advanced lung adenocarcinoma bearing no active driver mutations. Maintaining drug efficacy and overcoming drug resistance are essential. This study aimed to explore the real-world use of anti-diabetic agent metformin in combination with pemetrexed-based platinum doublets in a first-line setting. We retrospectively collected data during 2004~2013 from TaiwaN's National Health Insurance Research Database to access the survival benefit of metformin combined with pemetrexed-based platinum doublets as a first-line therapy for diabetic patients with advanced lung adenocarcinoma. Demographic data and information regarding platinum reagents, diabetes medications, and metformin doses were gathered, and overall survival status regarding metformin use was analyzed. Overall survival status based on the daily dose and the calculated cumulative defined daily dose (DDD) of metformin prescribed during the first 3 months after lung cancer was diagnosed was also assessed. A total of 495 patients were enrolled with a mean age of 67 years old, and the majority of the patients were male. After adjusting for age, sex, diabetes medication, and platinum reagents used, the adjusted hazard ratio (HR) for the metformin-user group was 0.61 (95% confidence interval (CI); 0.46~0.79; p < 0.001). The metformin-user group had a survival benefit (log-rank p < 0.001). We analyzed metformin dosing during the first 3 months after lung cancer diagnosis, and for a daily dose ≥ 1500 mg, the adjusted hazard ratio (aHR) was 0.42 (95% CI; 0.27~0.65; p < 0.001). Regarding the cumulative DDD of metformin, a DDD equal to or exceeding 21 resulted in aHR of 0.48 (95% CI; 0.34~0.69; p < 0.001). In this study, we found that the combination of metformin and pemetrexed-based platinum doublets provides a robust survival benefit as a first-line therapy for diabetic patients with advanced lung adenocarcinoma. It is worth conducting a large and randomized clinical trial to further investigate the antitumor effects of metformin on advanced lung adenocarcinoma when used as a first-ling therapy, including in non-diabetic patients.

Kefir Peptides Prevent Estrogen Deficiency-Induced Bone Loss and Modulate the Structure of the Gut Microbiota in Ovariectomized Mice.

Osteoporosis is a major skeletal disease associated with estrogen deficiency in postmenopausal women. Kefir-fermented peptides (KPs) are bioactive peptides with health-promoting benefits that are produced from the degradation of dairy milk proteins by the probiotic microflora in kefir grains. This study aimed to evaluate the effects of KPs on osteoporosis prevention and the modulation of the composition of the gut microbiota in ovariectomized (OVX) mice. OVX mice receiving an 8-week oral gavage of 100 mg of KPs and 100 mg of KPs + 10 mg Ca exhibited lower trabecular separation (Tb. Sp), and higher bone mineral density (BMD), trabecular number (Tb. N) and bone volume (BV/TV), than OVX groups receiving Ca alone and untreated mice, and these effects were also reflected in bones with better mechanical properties of strength and fracture toughness. The gut microbiota of the cecal contents was examined by 16S rDNA amplicon sequencing. α-Diversity analysis indicated that the gut microbiota of OVX mice was enriched more than that of sham mice, but the diversity was not changed significantly. Treatment with KPs caused increased microbiota richness and diversity in OVX mice compared with those in sham mice. The microbiota composition changed markedly in OVX mice compared with that in sham mice. Following the oral administration of KPs for 8 weeks, the abundances of Alloprevotella, Anaerostipes, Parasutterella, Romboutsia, Ruminococcus_1 and Streptococcus genera were restored to levels close to those in the sham group. However, the correlation of these bacterial populations with bone metabolism needs further investigation. Taken together, KPs prevent menopausal osteoporosis and mildly modulate the structure of the gut microbiota in OVX mice.

A mini-pig model for evaluating the efficacy of autologous platelet patches on induced acute full thickness wound healing.

BACKGROUND: Autologous platelet concentrates are currently widely used across different areas of regenerative medicine in order to enhance the wound healing process. Although several protocols for platelet concentrates are available, their application remains difficult due to different protocols leading to distinct products with vary potential biological uses. In this study, we attempted to make a platelet patch (PP) using mixtures of platelet rich plasma (PRP) injection and platelet rich fibrin (PRF) to promote wound repair and regeneration. RESULTS: Experiments were performed using a full-thickness wound model in mini-pigs. Autologous PRP, PRF and PP were prepared immediately before creating four full-thickness skin wounds in pigs. We quantified concentrations of platelets, thrombin and various growth factors to ensure that the desired effect can be produced. After surgery, hydrocolloid dressing, PRP injection, PRF and PP was applied to experimentally induced wounds. Application efficacy was evaluated by measurement of wound sizes and histological examination. The results indicated that all wounds showed a significant size reduction. Wound repair efficacy in response to PP treatment exhibited enhanced re-epithelialization compared to PRP and PRF (P < 0.05) and higher wound contraction than did PRF application (P < 0.05). Another aspect, experiment using DsRed transgenic pigs as blood donors demonstrated that leucocytes in PP were incorporated into the wound bed at the end of the study, suggesting that leucocytes activity is stimulated in response to PP application. Safety of the experimental processes was also confirmed by examination of organ biopsies. CONCLUSIONS: We used a mini-pig model to evaluate the efficacy of lab-made PP on induced full-thickness wound healing. Results demonstrated that application of one piece of PP was enough to obtain comparable efficacy versus general utilization of PRP or PRF for wound care. We also demonstrated that leucocytes in PP were incorporated into the wound bed and no safety concerns have been found in the whole experiment. This study provides a novel and feasible method for veterinary or clinical wound care.

Recombinant porcine myostatin propeptide generated by the Pichia pastoris elevates myoblast growth and ameliorates high-fat diet-induced glucose intolerance.

Myostatin (MSTN) was identified as a negative regulator of skeletal muscle growth. MSTN inhibition by myostatin propeptide (MSPP) increased skeletal muscle mass, myofiber growth and muscle force. Thus, this study was designed to produce wild-type porcine MSPP (WT-MSPP) and its mutated form (D75A-MSPP) in yeast Pichia pastoris and to investigate its potential enhancement of myoblast growth and differentiation. In an in vitro study, C2C12 myoblasts were treated with the purified WT-MSPP or D75A-MSPP (10 µg/mL) in either a regular culture medium or in a differentiation medium for 72 h. In an animal trial, post-weaning C57BL/6 mice fed with a high-fat diet (HFD) were administered WT-MSPP or D75A-MSPP for 6 weeks. The results showed that C2C12 myoblasts treated with the purified WT-MSPP or D75A-MSPP could dramatically promote cell proliferation. Both myoD and myogenin were significantly increased (p < .05) after WT-MSPP or D75A-MSPP treatment. D75A-MSPP was particularly more effective than WT-MSPP in promoting myotube formation (p < .05). The post-weaning mice treated with D75A-MSPP significantly increased both body and muscle weights compared with the mock and WT-MSPP groups (p < .05). Furthermore, the mice treatment with D75A-MSPP could prevent increased glucose injection from inducing glucose elevation. Our data indicated that a mutant-type MSPP (D75A-MSPP) was superior to WT-MSPP in effectively enhancing myofiber growth due to the highly resistant to proteolytic cleavage by the bone morphogenetic protein-1/tolloid (BMP-1/TLD) and thus has potential applications for clinical muscle wasting diseases or for increasing muscle mass in meat-producing animals.

Lactoferrin interacts with SPLUNC1 to attenuate lipopolysaccharide-induced inflammation of human nasal epithelial cells via down-regulated MEK1/2-MAPK signaling.

The short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein is an important innate material in the upper airway, and lactoferrin (LF) aids the innate functions in humans. In this study, a nasal epithelial model was used to investigate how LF modulates SPLUNC1 to reduce the inflammatory process mediated by lipopolysaccharide (LPS). The inflammation of human RPMI-2650 cells was induced with LPS to evaluate SPLUNC1 expression after treating the cells with bovine LF (bLF). The interaction pathway between LF and SPLUNC1 in LPS-induced inflammation was further investigated. Our study reveals that the addition of bLF results in the recovery of SPLUNC1 expression in nasal epithelial cells under LPS-induced inflammation. MAPK is involved in the main pathway for the SPLUNC1 and bLF interaction. Decreased SPLUNC1 function could be recovered by addition of bLF. The MEK1/2-MAPK signaling pathway is crucial for the SPLUNC1 and bLF interaction. Therefore, LF could support SPLUNC1 in the innate immunity recovery process.

A single amino acid in VP2 is critical for the attachment of infectious bursal disease subviral particles to immobilized metal ions and DF-1 cells.

VP2 protein is the primary host-protective immunogen of infectious bursal disease virus (IBDV). His249 and His253 are two surface histidine residues in IBDV subviral particles (SVP), which is formed by twenty VP2 trimers when the VP2 protein of a local isolate is expressed. Here, a systemic study was performed to investigate His249 or/and His253 on self-assembly, cell attachment and immunogenicity of SVP. Point-mutagenesis of either or both histidine residues to alanine did not affect self-assembly of the SVP, but the SVP lost its Ni-NTA binding affinity when the His253 was mutated. Indirect immunofluorescence assays and inhibitory experiments also showed that His253 is essential for SVP to attach onto the DF-1 cells and to inhibit IBDV infection of DF-1 cells. Finally, enzyme-linked immunosorbent assays and chicken protection assays demonstrated that SVP with a mutation of His253 to alanine induced comparable neutralizing antibody titers in chickens as the wild-type SVP did. It was concluded that VP2's His253, a site not significant for the overall immunogenicity induced by SVP, is crucial for the binding affinity of SVP to Ni-NTA and the attachment of an IBDV host cell line. This is the first paper to decipher the role of His253 played in receptor interaction and immunogenicity.

GDNF increases cell motility in human colon cancer through VEGF-VEGFR1 interaction.

Glial cell line-derived neurotrophic factor (GDNF), a potent neurotrophic factor, has been shown to affect cancer cell metastasis and invasion. However, the molecular mechanisms underlying GDNF-induced colon cancer cell migration remain unclear. GDNF is found to be positively correlated with malignancy in human colon cancer patients. The migratory activities of two human colon cancer cell lines, HCT116 and SW480, were found to be enhanced in the presence of human GDNF. The expression of vascular endothelial growth factor (VEGF) was also increased in response to GDNF stimulation, along with VEGF mRNA expression and transcriptional activity. The enhancement of GDNF-induced cancer cell migration was antagonized by a VEGF-neutralizing antibody. Our results also showed that the expression of VEGF receptor 1 (VEGFR1) was increased in response to GDNF stimulation, whereas GDNF-induced cancer cell migration was reduced by a VEGFR inhibitor. The GDNF-induced VEGF expression was regulated by the p38 and PI3K/Akt signaling pathways. Treatment with GDNF increased nuclear hypoxia-inducible factor 1 α (HIF1α) accumulation and its transcriptional activity in a time-dependent manner. Moreover, GDNF increased hypoxia responsive element (HRE)-containing VEGF promoter transcriptional activity but not that of the HRE-deletion VEGF promoter construct. Inhibition of HIF1α by a pharmacological inhibitor or dominant-negative mutant reduced the GDNF-induced migratory activity in human colon cancer cells. These results indicate that GDNF enhances the migration of colon cancer cells by increasing VEGF-VEGFR interaction, which is mainly regulated by the p38, PI3K/Akt, and HIF1α signaling pathways.

Endopeptidase activity characterization of E. coli-derived infectious bursal disease virus protein 4 tubules.

Viral protein 4 (VP4) is a serine protease that catalyzes the hydrolysis of polyprotein pVP2-VP4-VP3 of infectious bursal disease virus. In this report, the recombinant VP4 with a His-tag and three mutants (VP4-S652A, VP4-K692A and VP4-S652A.K692A) were expressed in Escherichia coli. Soluble VP4 was purified using immobilized metal-ion affinity chromatography or sucrose density gradient following with gel-filtration chromatography. The purified VP4 has a tubular structure with 25-30 nm in width and ∼300 nm in length, as observed by transmission electron microscope. A similar tubular structure was also found for these three mutants. The endopeptidase activity of these VP4 tubules was characterized by fluorescence resonance energy transfer using a synthetic fluorogenic oligopeptide as a substrate. The results show that the tubule-like VP4 is a functional enzyme with K(m) of 43 ± 2 µM and k(cat) of 0.04 ± 0.01 min⁻¹; however, k(cat) of three mutants were significantly reduced. This is the first report to demonstrate that VP4 protein expressed in E. coli can self-assemble into functional tubule-like particles and its activity can be completely inhibited by 1 mM of Ni⁺² ions.

Purification of capsid-like particles of infectious bursal disease virus (IBDV) VP2 expressed in E. coli with a metal-ion affinity membrane system.

Protein VP2, matured from the polyprotein encoded by the genome of infectious bursal disease virus (IBDV) and the primary host-protective immunogen of this virus, together with its two N-terminal truncated mutants were cloned and expressed in Escherichia coli. To obtain pure recombinant proteins for the development of an efficient subunit vaccine against IBDV infection, these three proteins were fused with six additional histidine residues at their C-termini as a His-purification-tag. Following purification employing immobilized metal-ion (Ni2+) affinity chromatography (IMAC), a purification fold of approximately 104 was achieved. Electron microscopic observation also demonstrated that all three E. coli-derived proteins form the morphology of icosahedral particles of approximately 25 nm in diameter. To reduce the cost of resin used for IMAC, self-prepared immobilized metal-ion affinity membranes (IMAM), i.e., commercial, regenerated cellulose membrane modified with iminodiacetic acid and immobilized with nickel ions, was applied to purify particles formed by these three proteins. A 104-fold of purification efficiency was also achieved by this membrane, showing that under the same conditions the recovery and purification efficiency of IMAM are comparable with those of IMAC. The pure VP2-formed particles thus obtained, coupled with their uniform dimensions, not only facilitate a better understanding of the structural biology of these immunogenic particles but also help the development of improved vaccines against this avian virus.