Two New Purification Methods of Hepatitis C Virus Particles from Serum-Free Culture System

Background: The traditional ultracentrifugation purification method of hepatitis C virus (HCV) particles requires special equipment, limiting its wide application. Therefore, more effective and convenient methods for HCV are needed. Objectives: The present study aimed to establish simple and effective purification methods for HCV. Methods: The infectious clone of the HCV genome (JFH-1) was transfected to the human hepatoma cell line (Huh7.5.1) and cultured in Dulbecco’s modified eagle medium/nutrient mixture F-12. The infectivity of JFH-1 culture was determined by reverse transcription-quantitative polymerase chain reaction and immunofluorescence. After concentration by centrifugal filter devices, HCV particles were purified by heparin-affinity chromatography and magnetic separation technique. The purified viruses were detected by the western blot and immune-electron microscopy. Results: The infectious titer of JFH-1 transfected Huh7.5.1 in the serum-free culture medium was 4.5 × 104 FFU/mL, and HCV ribonucleic acid load was 3.946 × 106 IU/mL in 30 days of cell culture post-transfection. After purification by heparin-affinity chromatography or magnetic separation method, viral particles were visualized with spherical morphology and an average diameter of 55 nm assessed by electron microscopy. The viruses were confirmed by the western blot and immune-electron microscopy with specific antibodies to HCV. Conclusions: The heparin-affinity chromatography and magnetic separation methods were established for the purification of HCV, which were simple and efficient methods for the stable purification of HCV particles on a large scale.

An Efficient Heparin Affinity Column Purification Method Coupled with Ultraperformance Liquid Chromatography for the Quantification of Native Lactoferrin in Breast Milk

Lactoferrin (LF) is a bioactive multifunctional protein and found in the highest amounts in human milk. Several methods can be used to quantify LF. However, quantification of native LF has garnered relatively little interest to date. This study aimed to develop a novel efficient two-step method for quantifying native LF in breast milk. During the analysis, LF was first extracted with phosphate buffer (pH 5.0), purified using a heparin affinity column. Subsequently, LF was detected using ultraperformance liquid chromatography (UPLC) at a wavelength of 201 nm. A linear calibration curve was obtained in the range of 5–200 mg/L. The limit of detection and limit of quantitation were 1 mg/L and 5 mg/L, respectively, indicating that the validated method could be employed to quantify LF in breast milk. Compared with previous HPLC methods, this method demonstrated several remarkable advantages, including simple operation, low-cost detection, and high accuracy. Hence, the results demonstrate an efficient method that can be employed commercially to purify and analyze LF in human milk samples.

Application of Heparin Affinity Chromatography to Produce a Differential Vaccine without Eliciting Antibodies against the Nonstructural Proteins of the Serotype O Foot-and-Mouth Disease Viruses

Although polyethylene glycol (PEG) application is the most widely used method in removing nonstructural proteins (NSPs) for foot-and-mouth disease (FMD) vaccine production, some NSPs remaining in the antigen could elicit antibodies against these proteins after repeated vaccinations in livestock. Therefore, the purpose of this study was to purify the FMD virus (FMDV) via affinity chromatography using a heparin ligand to remove most proteins, including NSPs. Chromatography showed an intact virus (146S) particle recovery of 70% or more for three different strains of serotype O FMDV (two locally isolated strains and one genetically modified strain). The experimental vaccine made with antigens eluted via heparin affinity chromatography elicited virus-neutralizing antibodies against homologous viruses but did not induce antibodies against NSPs even after five immunizations in goats; this indicated that the NSPs were effectively removed from the vaccine antigen. This method can then be used to produce a higher-quality vaccine compared with PEG application in terms of the purity of the FMD vaccine. Therefore, this result would be an important groundwork for advanced FMD vaccine manufacturing in the near future.

Heterologous expression of human KGF/FGF7 (Keratinocyte growth factor) in Pichia pastoris

Keratinocyte Growth Factor (KGF) is a paracrine-acting and epithelium-specific growth factor produced by cells of mesenchymal origin, play an important role in promoting proliferation, differentiation, motility of epithelial cells and stimulating regeneration of damaged epithelial tissues. Recent studies indicated that recombinant KGF is produced in many different expression systems such as bacteria, insect cells, plant and mammalian cells. However, KGF’s yields obtained from these systems is low and production’s cost is high especially in mammalian cells. In this study, the yeast Pichia pastoris was chosen as a host for KGF expression through induction of methanol by promoter AOX on pPICzαA vector system. The results demonstrated that the Pichia pastoris X33:kgf transformants secreted KGF directly into BMMY medium after inducing by 0.5% methanol. The recombinant protein was purified by heparin affinity chromatography with the yield of 1.35 mg/l and the purity of 99.89% showed by SDS-PAGE. In addition, MTT assay showed the purified recombinant KGF had a proliferation effect on A549 cell line since A549 known as a cell has KGF’s receptor.

NFAT5 regulates renal gene expression in response to angiotensin II through Annexin-A2-mediated posttranscriptional regulation in hypertensive rats

The aim was to identify new targets that regulate gene expression at the posttranscriptional level in angiotensin II (ANGII)-mediated hypertension. Heparin affinity chromatography was used to enrich nucleic acid-binding proteins from kidneys of two-kidney, one-clip (2K1C) hypertensive Wistar rats. The experiment was repeated with 14-day ANGII infusion using Alzet osmotic mini pumps, with or without ANGII receptor AT1a inhibition using losartan in the drinking water. Mean arterial pressure increased after 2K1C or ANGII infusion and was inhibited with losartan. Heparin affinity chromatography and mass spectrometry were used to identify Annexin-A2 (ANXA2) as having differential nucleic acid-binding activity. Total Annexin-A2 protein expression was unchanged, whereas nucleic acid-binding activity was increased in both kidneys of 2K1C and after ANGII infusion through AT1a stimulation. Costaining of Annexin-A2 with α-smooth muscle actin and aquaporin 2 showed prominent expression in the endothelia of larger arteries and the cells of the inner medullary collecting duct. The nuclear factor of activated T cells (NFAT) transcription factor was identified as a likely Annexin-A2 target using enrichment analysis on a 2K1C microarray data set and identifying several binding sites in the regulatory region of the mRNA. Expression analysis showed that ANGII increases NFAT5 protein but not mRNA level and, thus, indicated that NFAT5 is regulated by posttranscriptional regulation, which correlates with activation of the RNA-binding protein Annexin-A2. In conclusion, we show that ANGII increases Annexin-A2 nucleic acid-binding activity that correlates with elevated protein levels of the NFAT5 transcription factor. NFAT signaling appears to be a major contributor to renal gene regulation in high-renin states.

Determination of Bovine Lactoferrin in Food by HPLC with a Heparin Affinity Column for Sample Preparation

An HPLC method was developed for the quantitative determination of bovine lactoferrin (bLF) in sterilized milk, modified milk, fermented milk, infant formula, adult formula, rice cereal, vitamin function drink, and protein powder products. bLF was first extracted with a phosphate buffer (pH 8), underwent cleanup in a heparin affinity column, and was detected by HPLC with a C4 column and diode-array detector at a wavelengthof 280 nm. The proposed method provided a linear detection range of 10.0–1000 μg/mL with an LOD of 0.6 mg/100 g in liquid samples and 3 mg/100 g in solid samples and an LOQ of 2 mg/100 g in liquid samples and 10 mg/100 g in solid samples. In addition, the method showed good recovery for various samples, ranging from 76 to 96%. The method had several remarkable advantages, including ease of handling, high sensitivity and accuracy, good reproducibility, and low-cost detection. Based on the distinctive properties presented here, webelieve the proposed HPLC assay holds great promise for the oversight and detection of bLF in testing organizations, dairy enterprises, and regulatory authorities.