Prokaryotic expression, refolding, and purification of functional human vascular endothelial growth factor isoform 165: purification procedures and refolding conditions revisited.

Human vascular endothelial growth factor isoform 165 (VEGF165) is the first known member belonging to the VEGF protein family that plays a critical role in new blood vessel formation in vivo. This study presents a new protocol with optimized conditions for rapidly producing untagged recombinant and biological active human VEGF165 (rhVEGF165) using Escherichia coli cells. Protein was isolated from inclusion bodies, purified by gel filtration and ion exchange chromatography, and subjected to protein refolding and renaturation. The biological activity of rhVEGF165 is comparable with VEFG from eukaryotic source according to human umbilical vein endothelial cells (HUVEC) proliferation assay. Therefore, the present procedures provide a fast and easy way to produce this therapeutic protein.

Identification of in vivo phosphorylation sites of lens proteins from porcine eye lenses by a gel-free phosphoproteomics approach.

PURPOSE: Phosphorylation is an important post-translational modification for the cellular regulation of various biosignaling pathways. We have identified in vivo phosphorylation sites of various lens proteins including especially the major structural proteins of the crystallin family from porcine eye lenses by means of two-dimensional gel electrophoresis (2-DE) or immobilized metal affinity chromatography (IMAC) followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). METHODS: For the identification of phosphorylated residues in various lens proteins of porcine lens extracts, we have adapted two complementary proteomic approaches, i.e., pre-fractionation of protein samples with 2-DE or enrichment of phosphopeptides with IMAC followed by LC-MS/MS analysis and database search. The results were compared and validated with those in phosphoproteomics databases. RESULTS: Two subunits of alpha-crystallin, alphaA-crystallin and alphaB-crystallin, as well as other lens crystallins and non-crystallin cellular proteins, such as beta-enolase, heat shock protein beta-1 (HSP27), and glucose-6-phosphate isomerase (GPI) were found to be phosphorylated in vivo at specific sites. Moreover, alphaA- and alphaB-crystallins were found to be the most abundantly phosphorylated proteins in porcine lenses, being extensively phosphorylated on serine or threonine, but not on tyrosine residues. CONCLUSIONS: The complementary gel-based and gel-free proteomic strategies have been compared and evaluated for the study of crystallin phosphorylation from whole tissue extracts of porcine eye lenses. Technically, the IMAC method facilitates direct site-specific identification of phosphorylation residues in lens proteins, which does not necessitate the pre-MS/MS 2-DE separation of protein samples. Moreover, the improved strategy using gel-free phosphoproteomics analysis affords a more effective and simplistic method for the determination of in vivo phosphorylation sites than the conventional 2-DE pre-separation of protein mixture. This study should form a firm basis for the comprehensive analysis of post-translational modification of lens proteins in terms of aging or various diseased states.

A novel albumin-based tissue scaffold for autogenic tissue engineering applications.

Tissue scaffolds provide a framework for living tissue regeneration. However, traditional tissue scaffolds are exogenous, composed of metals, ceramics, polymers, and animal tissues, and have a defined biocompatibility and application. This study presents a new method for obtaining a tissue scaffold from blood albumin, the major protein in mammalian blood. Human, bovine, and porcine albumin was polymerised into albumin polymers by microbial transglutaminase and was then cast by freeze-drying-based moulding to form albumin tissue scaffolds. Scanning electron microscopy and material testing analyses revealed that the albumin tissue scaffold possesses an extremely porous structure, moderate mechanical strength, and resilience. Using a culture of human mesenchymal stem cells (MSCs) as a model, we showed that MSCs can be seeded and grown in the albumin tissue scaffold. Furthermore, the albumin tissue scaffold can support the long-term osteogenic differentiation of MSCs. These results show that the albumin tissue scaffold exhibits favourable material properties and good compatibility with cells. We propose that this novel tissue scaffold can satisfy essential needs in tissue engineering as a general-purpose substrate. The use of this scaffold could lead to the development of new methods of artificial fabrication of autogenic tissue substitutes.

ΑB-crystallin in clear cell renal cell carcinoma: tumor progression and prognostic significance.

OBJECTIVES: AlphaB-crystallin (αB-crystallin), a small heat shock protein, has been reported to be involved in the growth, antiapoptosis, migration, and chemoresistance of human malignancies. MATERIALS AND METHODS: αB-crystallin expression in normal renal and clear cell renal cell carcinoma (ccRCC) tissues was examined with two-dimensional (2D) gel electrophoresis assays. Immunohistochemistry was conducted to determine the presence of αB-crystallin-positive tumor cells and staining intensity in 50 cases of ccRCC tissue samples. The association of αB-crystallin protein expression, clinicopathogic parameters and prognosis of ccRCC patients was also analyzed with Student's t-test and Kaplan-Meier analysis. Moreover, Western blot assays were performed to detect the protein expression of αB-crystallin in normal and tumor tissues and the alteration of cell cycle regulators in αB-crystallin-overexpressing cells. MTT (3-[4,5-dimethythiazol-2-yl]-2,5-diphenyltetrazolium bromide), BrdU, and transwell assays were performed to demonstrate the effects of αB-crystallin overexpression on cell growth, DNA synthesis and cell migration of ccRCC cells, respectively. RESULTS: The results showed the up-regulation of αB-crystallin expression in ccRCC tissues. Overall survival of ccRCC patients was significantly correlated with αB-crystallin expression in tumor tissues. We found that αB-crystallin overexpression increased the expression of cyclin A and the incorporation of BrdU, which may be related to the enhancement of cell growth. Transwell analyses demonstrated that presence of αB-crystallin overexpression enhanced cell migration in ccRCC cells. Furthermore, rapamycin-resistance of tumor cells was induced when αB-crystallin was overexpressed. CONCLUSIONS: Our experimental findings highlight the importance of αB-crystallin in the tumor growth, migration, and target therapy-resistance of ccRCC cells.

Synthesis and interfacing of biocompatible iron oxide nanoparticles through the ferroxidase activity of Helicobacter Pylori ferritin.

Ferritin is an iron storage protein that is often used to coat metallic nanoparticles, such as iron oxide nanoparticles (IONPs). However, the synthesis and biocompatibility of ferritin-coated IONPs remain unclear. Therefore, this study reports the synthesis of a ferritin gene cloned and expressed from Helicobacter pylori (HPFn). The ferroxidase activity of the synthase HPFn was used for the de novo synthesis of HPFn-coated IONPs under mild conditions. Gel filtration chromatography and transmission electron microscopy analyses demonstrated that the core-shell structure of both the 5.0 nm IONP nanocore and the 12.4 nm HPFn shell were correctly assembled. The cellular uptake of mouse macrophage cells (RAW 264.7 cells) has shown that only a few HPFn-coated IONPs (3%) were taken up after 24 h of incubation. This study compares the biocompatibility of HPFn-coated IONPs, superparamagnetic iron oxide nanoparticles (SPIOs) and ferric salt (ferric ammonium citrate) in respect to cell growth inhibition, reactive oxygen species generation and pro-inflammatory cytokine TNF-α release. Assessment results showed that the responses elicited by HPFn-coated IONPs were similar to those elicited by SPIO treatment but milder than those elicited by ferric salt treatment. This accounts for the notion that ferritin-coated IONPs are biocompatible iron agents. These findings show that the ferroxidase activity of ferritin can be used to synthesize biocompatible IONPs. The favorable properties of HPFn-coated IONPs suggest that they can be used as a non-macrophage contrast agent through further surface conjugation.

Identifying the regulatory element for human angiotensin-converting enzyme 2 (ACE2) expression in human cardiofibroblasts.

Angiotensin-converting enzyme 2 (ACE2) has been proposed as a potential target for cardioprotection in regulating cardiovascular functions, owing to its key role in the formation of the vasoprotective peptides angiotensin-(1-7) from angiotensin II (Ang II). The regulatory mechanism of ace2 expression, however, remains to be explored. In this study, we investigated the regulatory element within the upstream of ace2. The human ace2 promoter region, from position -2069 to +20, was cloned and a series of upstream deletion mutants were constructed and cloned into a luciferase reporter vector. The reporter luciferase activity was analyzed by transient transfection of the constructs into human cardiofibroblasts (HCFs) and an activating domain was identified in the -516/-481 region. Deletion or reversal of this domain within ace2 resulted in a significant decrease in promoter activity. The nuclear proteins isolated from the HCFs formed a DNA-protein complex with double stranded oligonucleotides of the -516/-481 domain, as detected by electrophoretic mobility shift assay. Site-directed mutagenesis of this region identified a putative protein binding domain and a potential binding site, ATTTGGA, homologous to that of an Ikaros binding domain. This regulatory element was responsible for Ang II stimulation via the Ang II-Ang II type-1 receptor (AT1R) signaling pathway, but was not responsible for pro-inflammatory cytokines TGF-ß1 and TNF-α. Our results suggest that the nucleotide sequences -516/-481 of human ace2 may be a binding domain for an as yet unidentified regulatory factor(s) that regulates ace2 expression and is associated with Ang II stimulation.

Upregulation of a non-heme iron-containing ferritin with dual ferroxidase and DNA-binding activities in Helicobacter pylori under acid stress.

Helicobacter pylori is a spiral Gram-negative microaerophilic bacterium. It is unique and distinctive among various bacterial pathogens for its ability to persist in the extreme acidic environment of human stomachs. To address and identify changes in the proteome of H. pylori in response to low pH, we have used a proteomic approach to study the protein expression of H. pylori under neutral (pH 7) and acidic (pH 5) conditions. Global protein-expression profiles of H. pylori under acid stress were analysed by two-dimensional polyacrylamide gel electrophoresis (2-DE) followed by liquid chromatography (LC)-nanoESI-mass spectrometry (MS)/MS and bioinformatics database analysis. Among the proteins differentially expressed under acidic condition, a non-heme iron-containing ferritin of H. pylori (HP-ferritin) was found to be consistently upregulated at pH 5 as compared to pH 7. It was also found that HP-ferritin can switch from an iron-storage protein with ferroxidase activity to a DNA-binding/protection function under in vitro conditions upon exposure to acidic environment. Prokaryotic ferritins, such as non-heme iron-binding HP-ferritin with dual functionality reported herein, may play a significant urease-independent role in the acid adaptation of H. pylori under physiological conditions in vivo.