Dexian Dong

Affinity purification of egg yolk immunoglobulins (IgY) with a stable synthetic ligand

Chicken IgY (egg yolk immunoglobulin) is a functional equivalent of mammalian IgG. Traditional methods for IgY purification involve multi-step procedures that result in low recovery of IgY. After a large scale screening of our 700-member synthetic ligand library synthesized by epichlorohydrin and cyanuric chloride methods, a high efficiency ligand of IgY was found. By one-step purification with this ligand, the purity of IgY could reach 92.1%, and the recovery of IgY could reach 78.2%. This synthetic ligand had a higher binding capacity of 74.8 mg IgY/ml and had no negative effects on immunoreactivity. Remarkably, this ligand was also highly stable and could resist 1M NaOH, thus having great potential for the industrial-scale production of IgY.

Phenazine-1-carboxylic acid derivatives: design, synthesis and biological evaluation against Rhizoctonia solani Kuhn.

Rhizoctonia solani Kuhn is the pathogen that causes sheath blight and results in significant yield reduction in rice and in nearly 50 other crops. In order to develop a new fungicide effective against this pathogen, a series of structurally diverse phenazine-1-carboxylic acid derivatives, 2a, 2b, 2c, 2d, 2e, 2f, 2g, 2h, 2i, 2j, and 2k, were designed, synthesized and evaluated for their antifungal activity. The two most active compounds 2i and 2j were selected as lead compounds for further antifungal research.

Biomimetic affinity purification of cardiotoxin and its pharmacological effects on the nervous system

Cobra venom is a very precious natural resource. The traditional method for purification of cardiotoxin from cobra venom is a multi‐step, high cost, and low recovery procedure. By molecular modeling and docking with SYBYL software, we designed and synthesized an affinity ligand, m‐aminobenzoic acid, for high efficiency purification of this therapeutically useful Chinese cobra venom cardiotoxin. The one‐step recovery of cardiotoxin reached 64% and the purity reached 92% upon purification. The binding capacity of this synthetic ligand was 9.1 mg cardiotoxin/g moist weight gel and the affinity constant for cardiotoxin was 5.5 × 103 M−1. Unlike a natural affinity ligand, this synthetic ligand is highly stable, and has great potential for industrial scale production of cardiotoxin. In addition, we examined the effects of cardiotoxin on the nervous system in a mouse model. Results showed that cardiotoxin could maintain analgesic effects for 120 min with a dose of less than 0.06 mg/kg (2.8% of the LD50). Administration of 0.12 mg/kg cardiotoxin could improve scopolamine impairments of memory in mice. These results suggest that cardiotoxin may be a potential drug for nervous system diseases. Copyright

Utilizing a library of synthetic affinity ligands for the enrichment, depletion and one-step purification of leech proteins

Although the concept of affinity purification using synthetic ligands had been utilized for many years, there are few articles related to this research area, and they focus only on the affinity purification of specific protein by a defined library of synthetic ligands. This study presents the design and construction of a 700‐member library of synthetic ligands in detail. We selected 297 ligand columns from a 700‐member library of synthetic ligands to screen leech protein extract. Of the 297, 154 columns had an enrichment effect, 83 columns had a depletion effect, 36 columns had a one‐step purification effect, and 58 columns had a one‐step purification via flowthrough effect. The experimental results achieved by this large library of affinity ligands provide solid convincing data for the theory that affinity chromatography could be used for the enrichment of proteins that are present in low abundance, the depletion of high abundance proteins, and one‐step purification of special proteins. Copyright

Pre‐fractionation of rat liver cytosol proteins prior to mass spectrometry‐based proteomic analysis using tandem biomimetic affinity chromatography

Efficient and high resolution separation of the protein mixture prior to trypsin digestion and mass spectrometry (MS) analysis is generally used to reduce the complexity of samples, an approach that highly increases the probability of detecting low‐copy‐number proteins. Our laboratory has constructed an affinity ligand library composed of thousands of ligands with different protein absorbance effects. Structural differences between these ligands result in different non‐bonded protein–ligand interactions, thus each ligand exhibits a specific affinity to some protein groups. In this work, we first selected out several synthetic affinity ligands showing large band distribution differences in proteins absorbance profiles, and a tandem composition of these affinity ligands was used to distribute complex rat liver cytosol into simple subgroups. Ultimately, all the fractions collected from tandem affinity pre‐fractionation were digested and then analyzed by LC‐MS/MS, which resulted in high confidence identification of 665 unique rat protein groups, 1.8 times as many proteins as were detected in the un‐fractionated sample (371 protein groups). Of these, 375 new proteins were identified in tandem fractions, and most of the proteins identified in un‐fractionated sample (290, 80%) also emerged in tandem fractions. Most importantly, 430 unique proteins (64.7%) only characterized in specific fractions, indicating that the crude tissue extract was well distributed by tandem affinity fractionation. All detected proteins were bioinformatically annotated according to their physicochemical characteristics (such as MW, pI, GRAVY value, TM Helices). This approach highlighted the sensitivity of this method to a wide variety of protein classes. Combined usage of tandem affinity pre‐fractionation with MS‐based proteomic analysis is simple, low‐cost, and effective, providing the prospect of broad application in proteomics. Copyright

A novel fractionation method prior to MS-based proteomics analysis using cascade biomimetic affinity chromatography.

This is the first report that combines cascade biomimetic affinity fractionation with MS-based proteomics analysis. Our lab has constructed an affinity ligand library composed of thousands of ligands with different protein-binding properties. Structural differences between these ligands result in different non-bonded protein-ligand interactions, thus each ligand exhibits a specific affinity to some protein groups. In this work, we first screened out three affinity ligands with large difference in protein-binding properties. Next, cascade combination of these ligands was applied to fractionate tissue sample into simple subgroups prior to trypsin digestion and LC-MS/MS analysis. In this study, 391 non-redundant protein groups were identified in unfractionated rat liver cytosol, 499 protein groups were identified in 2 fractions of the first affinity fractionation, 616 in 4 fractions of the second fractionation, and 738 in 8 fractions of the third fractionation (an 88.74% increase). Ultimately, a total of 859 unique protein groups were identified in all cascade fractions (a 119.6% increase compared with unfractionated sample). The proteins detected in each fraction were bioinformatically categorized according to their physicochemical characteristics (relative molecular mass, pI, GRAVY value and TM helices). This approach highlighted the sensitivity of this method to a wide variety of protein classes.

Structural and biological characterization of a novel acutobin‐like enzyme isolated from the venom of the sharp‐nosed pit viper (Deinagkistrodon acutus)

We previously reported the purification of a serine proteinase from the venom of the sharp‐nosed pit viper (Deinagkistrodon acutus) using a combination of affinity chromatography and ion‐exchange chromatography [Xin, Dong, Wang and Li, R. (2007) J. Chromatogr. B 859, 111–118]. The high fibrinogen‐clotting activity [2025 NIH (National Institutes of Health) units/mg] of this protein indicated that it may have great potential as a drug for treating thrombolysis. In order to systemically determine the purified proteins structure and activity, it was characterized using the following methods: MS, isoelectric focusing, deglycosylation analysis, amino acid composition analysis, peptide mass fingerprinting, N‐terminal amino acid sequencing, CD, hydrophobic‐site analysis and bioactivity assays. In addition, a fluorescence probe was synthesized and conjugated to the protein in order to analyse its active site. The results indicated that the protein is a novel acutobin‐like enzyme (designated acutobin II) with strong clotting and esterase activities and is composed of a 28 kDa peptide chain plus approx. 6 kDa of O‐linked glycan chains. The protein contains 249 amino acids and, remarkably, no tryptophan residues. The pI of the protein is 4.8±0.2. The proteins secondary structure is dominated by β‐sheets (49%) and random coils (43%), and its tertiary structure does not contain any metal ions or disulfide bonds and possesses only one hydrophobic pocket. Analysis revealed that the hydrophobic pocket is most likely the enzymatic active site.

Combined usage of cascade affinity fractionation and LC‐MS/MS for the proteomics of adult mouse testis

In this report, the proteomics of adult mouse testis were analyzed by the combined usage of cascade affinity fractionation and LC-MS/MS. The differences between the selected affinity ligands in size, shape, structure, and biochemical characteristics, result in each ligand exhibiting a specific affinity to some protein groups. Therefore, a cascade composition of different ligands can be applied to the fractionation of complex tissue proteins. Ultimately, the fractions collected from cascade affinity fractionation were analyzed by LC-MS/MS, which resulted in high confidence identification of a total of 1378 non-redundant mouse testis protein groups, over 2.6 times as many proteins as were detected in the un-fractionated sample (526). All detected proteins were bioinformatically categorized according to their physicochemical characteristics (such as relative molecular mass, pI, grand average hydrophobicity value, and transmembrane helices), subcellular location, and function annotation. This approach highlighted the sensitivity of this method to a wide variety of protein classes. Utilizing a combination of cascade affinity fractionation and LC-MS/MS, we have established the largest proteomic database for adult mouse testis at the present time.

Utilizing RNA/DNA hybridization to directly quantify mRNA levels in microbial fermentation samples

mRNA quantification has become a research hotspot. Quantitative real-time RT-PCR is a popular method but is known to lack precision. To rapidly monitor the kinetics of mRNA levels for the control of microbial fermentation processes, we developed an SYBR Green I-based universal method to directly quantify mRNA from fermentation samples. After total RNA was extracted, the mRNA was hybridized and protected by a longer DNA oligonucleotide. The probe length determined the strength of signal amplification. S1 nuclease and RNase A were used to remove excess probe, single-stranded RNA, and mis-matched RNA/DNA hybrids. Finally, the perfect-matched RNA/DNA hybrid was quantified by SYBR Green I dye. The conditions of liquid hybridization and enzyme digestion were systemically optimized. The kinetic tendency of phzC mRNA levels during phenazine-1-carboxylic acid fermentation was consistent with the results from MB hybridization in our previous report. The detection of mRNA levels of ten genes in Pseudomonas sp. M18G proved that this method is universal and feasible for mRNA quantification, and has great potential for application in mRNA quantification in various organisms.

An easily-automated assay for the physiological state quantification of Pseudomonas sp. M18.

In order to foreknow poorly performing cultures before wasting energy to scale them to large cultures, industrial microbial fermentation can greatly benefit from knowledge of the physiological state of cells. The method currently proposed is an easily automated physiological state determination method. We have designed one universal rRNA-specific probe for bacteria and developed novel signal probe hybridization (SPH) assay featuring no RNA extraction and no PCR amplification steps necessary to quantify the physiological state of microbial cells. The microbial cell was lysed with sonication and SDS. Signal probes were applied to hybridize and protect the rRNA target. S1 nuclease was then applied to remove the excessive signal probes, the single-stranded RNA and the mismatch RNA/DNA hybrids. The remaining signal probe was captured with a corresponding capture probe immobilized on a microplate and quantified with a horseradish peroxidase-conjugated color reaction. We then systemically optimized our assay. Results showed that the cell limit of detection (LOD) and the cell limit of quantification (LOQ) were 2.64 x 10(4) cells and 9.86 x 10(4) cells per well of microplate, respectively. The limit of detection (LOD) and the limit of quantification (LOQ) of signal probe were 49.0 fM and 344.0 fM respectively. Using this technique, we quantified the 16S rRNA levels during the fermentation process of Pseudomonas sp. M18. Our results indicate that the 16S rRNA levels can directly inform us about the physiological state of microbial cells. This technique has great potential for application to the microbial fermentation industry.