Abstract:Polymalic acid, known as a bioactive material, is completely biodegradable, and has far reaching application potential in medical field. Combined with our own findings, we summarized advances in polymalic acid metabolism, microbial fermentation synthesis, and application research in the medical field. Finally, prospect for further research was addressed.

Abstract:As the major pathway mediating specific protein degradation in eukaryotes, ubiquitin-proteasome system (UPS) is involved in various physiological and pathological processes such as cell cycle regulation, immune response, signal transduction and DNA-repair. Deubiquitinases (DUB) maintain the balance of UPS and related physiological processes via reversibly removing ubiquitin from the covalently modified protein substrates, which have been implicated in various disease processes in case of their imbalance expression. Because DUB plays critical regulating roles in the UPS pathway, they may be also the ideal drug targets for severe and intractable human diseases, such as cancer and neurodegenerative disease. With the rapid development of proteomic technology, systematical investigation of specific substrates and interacting proteins of varied DUB via mass spectrometry approach may shed light on these DUB’s biological function and regulating roles in the physiological and pathogenic states. In this review, we briefly introduce the characteristics of DUB and summarize the recent application and progresses of proteomics in DUB research.

Abstract:Nanobodies are derived from the vari-able domain of the heavy-chain antibodies (HCAbs) that occur naturally in the serum of Camelidae. They are the smallest antibody fragments capable to bind antigens. With the characteristics of their increased solubility, increased domain stabilities, nanomolar affinities, easy crossing the blood-brain barrier, easy generation, engineering, optimization and tailoring, easy humanization, nanobodies have extensive application prospects in diagnosis and detection. Although nanobody has demonstrated tremendous success, a number of practical challenges limit its broader applications in disease diagnosis and detection, including construction of a phage library and selection of nanobody fragments with high affinity and immunogold labeling technique. Here, we review several recent findings on the use of nanobodies in molecular diagnostics and suggest some practical strategies in resolving the current challenges in this attractive research area, particularly to optimize the affinity, solubility, humanization of nanobodies.

Abstract:Determination of protein 3-dimensional structure offers very important information in biology researches, especially for understanding protein functions and redundant drug design. The X-ray crystallography is still the main technique for protein structure determination. Obtaining protein crystals is an essential procedure after protein purification in this technique. However, there is only 42% of soluble purified proteins yield crystals by statistics. Experimental verification of protein crystallizability is relatively expensive and time-consuming. Thus it is desired to predict the protein crystallizability by a computational method before starting the experiment. In this paper, combined with our own efforts, some successful in silico methods to predict the protein crystallizability are reviewed.

Abstract:The coding sequence for the mature peptide of porcine lactoferrin (Plf) was synthesized according to the codon usage of lactobacillus, to establish optimized porcine lactoferrin Lactobacillus expression system. The gene was ligated into the XhoⅠ/BamHⅠsite of Lactobacillus expression vector pPG612.1 and the recombinant plasmid pPG612.1-plf was transformed individually into Lactobacillus casei ATCC393, Lactobacillus pentosus KLDS1.0413, Lactobacillus plantarum KLDS1.0344 or Lactobacillus paracasei KLDS1.0652 by electroporation. After induction with xylose, expression of the recombinant proteins was detected by Western blotting and confocal laser scanning microscopy. Secretion of recombinant Plf proteins from four recombinant species was determined quantitatively by ELISA. The antibacterial activities of recombinant proteins were measured by agar diffusion method. The result shows that Plf was correctly expressed in four species of recombinant lactobacillus, with molecular weight of about 73 kDa. The expression levels in recombinant Lactobacillus casei, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus paracasei were 9.6 μg/mL, 10.8 μg/mL, 12.5 μg/mL and 9.9 μg/mL, respectively. Antimicrobial activity experiment shows that the recombinant proteins were active against E. coli, Staphylococcus aureus, Salmonella typhimurium, Listeria, Pasteurella. The recombinant Plf expressed by recombinant Lactobacillus plantarum showed the best antibacterial activity among all recombinant lactobacillus species. These data represent a basis for the development and application of porcine lactoferrin from recombinant lactobacillus.

Abstract:Glycerol is the main byproduct in ethanol production by Saccharomyces cerevisiae. In order to improve ethanol yield and the substrate conversion, a cassette about 4.5 kb for gene homologous recombination, gpd2Δ::PGK1PT-POS5-HyBR, was constructed and transformed into the haploid strain S. cerevisiae S1 (MATa) to replace the GPD2 gene by POS5 gene. The NADH kinase gene POS5 was successfully expressed in the recombinant strain S. cerevisiae S3. Comparing with the parent strain, the recombinant strain S. cerevisiae S3 exhibited an 8% increase in ethanol production and a 33.64% decrease in glycerol production in the conical flask fermentation with an initiatory glucose concentration of 150 g/L. Overexpression of NADH kinase gene seems effective in reducing glycerol production and increasing ethanol yield.

Abstract:4″-O-isovaleryltransferase gene (ist) was regulated by positive regulatory genes of midecamycin 4″-O-propionyltransferase gene (mpt) in Streptomyces lividans TK24. A BamHⅠ~8.0 kb fragment from Streptomyces mycarofaciens 1748 was proved that it contained mpt gene and linked with two positive regulatory genes, orf27 and orf28. Orf of mpt was replaced by orf of ist and linked with two regulatory genes or orf27 single, and individually cloned into the vectors pKC1139 or pWHM3 (high copy number), and then transformed into S. lividans TK24. The levels of mpt and ist expression were evaluated by the bio-tramsformation efficacy of spiramycin into 4″-O-acylspiramycins in these transformants. The results showed that 4"-O-isovalerylspiramycins could be detected only in the transformants containing the plasmids constructed with pWHM3. The efficacy of bio-transformation of the transformants containing two regulatory genes was higher than that of orf27 single. So, the positive regulatory genes system of mpt gene could enhance ist gene expression.

Abstract:In this study, we constructed a yeast consortium surface-display expression system by using Flo1 as an anchor protein. Endoglucanase II (EGII) and cellobiohydrolase II (CBHII) from Trichoderma reesei, and β-glucosidase 1 (BGLI) from Aspergillus aculeatus were immobilized on Saccharomyces cerevisiae Y5. We constructed the cellulose-displaying expression yeast consortium (Y5/fEGII:Y5/fCBHII:Y5/fBGLI=1:1:1) and investigated the enzymatic ability and ethanol fermentation. The displayed cellulolytic enzymes was stabile during the 96-h fermentation. The yeast consortium produced 0.77 g/L ethanol from 10 g/L phosphoric acid?swollen cellulose (PASC) within 96 h. The yield (in grams of ethanol produced per gram of carbohydrate consumed) was 0.35 g/g, which correspond to 68.6% of the theoretical yield.

Abstract:Fatty acid synthase (FAS) catalyses the reaction between acetyl-CoA and malonyl-CoA to produce fatty acids, an important enzyme in lipid biosynthesis. FAS of the oleaginous yeast Rhodosporidium toruloides has two acyl carrier protein (ACP) domains and a distinct subunit composition compared with FASs of other species. As ACP is a protein cofactor crucial for fatty acid chain elongation, more ACPs in the FAS may facilitate the reaction. To study the biochemical and structural properties of this novel FAS from R. toruloides, plasmids were constructed and transformed into Escherichia coli BL21(DE3). The strain ZWE06 harboring plasmids pET22b-FAS1 and pET24b-FAS2 could co-overexpress the two subunits. The recombinant FAS was purified by sequentially using ammonium sulphate precipitation, sucrose density gradient centrifugation and anion exchange chromatography. The specific activity of the recombinant FAS was 548 mU/mg. The purified complex would be used to study enzyme kinetics and protein structure of FAS, and heterogeneous expression and purification will facilitate revealing the mechanism of this novel FAS with double ACPs.

Abstract:We analyzed the whole genome coding sequence of Volvariella volvacea to study the pattern utilization of codons by Codon W 1.4.2. As results, 24 optimal codons were identified. Moreover, the frequency of codons usage was calculated by CUSP program. We compared the frequency of codons usage of V. volvacea with other organisms including 6 modal value species (Homo sapiens, Saccharomys cerevisiae, Arabidopsis thalian, Mus musculus, Danio rerio and Drosophila melanogaster) and 4 edible fungi (Coprinopsis cinerea, Agaricus bisporus, Lentinula edodes and Pleurotus ostreatus). We found that there were less differences in 3 edible fungi (excluding Pleurotus ostreatus) than 6 modal value species, comparing with the frequency of codons usage of V. volvacea. With software SPSS16.0, cluster analysis which showed differences in the size of codon bias, reflects the evolutionary relationships between species, which can be used as a reference of evolutionary relationships of species. This was the first time for analysis the codon preference among the whole coding sequences of edible fungi, serving as theoretical basis to apply genetic engineering of V. volvacea.

Abstract:Sorting nexins (SNXs) are a large group of proteins that contain Phox (PX)domain and involve in regulating endocytosis and endosome sorting. SNX7, a member of SNXs family, contains a PX domain and a BAR domain. In zebrafish, SNX7 is a liver-enriched anti-apoptotic protein and indispensible for the liver development. A fragment of SNX7 cDNA (px-barsnx7), encoding the PX domain and the BAR domain, was inserted into the expressing vector p28a, transformed into E. coli Rosseta 2 (DE3), and then induced by isopropyl β-D-1-Thiogalactopyranoside (IPTG). After affinity, ion exchange and gel filtration purification, the purity of PX-BARSNX7 reached over 95%. Dynamic light scattering (DLS) experiment indicated that PX-BARSNX7 was homogeneous in solution. Lipid overlay assay showed that PX-BARSNX7 can bind to PtdIns(5)P, PtdIns(4,5)P2 and PtdIns(3,4,5)P3.

Abstract:In proteomic research, to improve protein solubility of membrane proteins and nuclear proteins, buffers containing high concentration of detergent, such as 4% SDS, were widely used. However, high concentration of detergent might severely interfere with the downstream proteomic analysis, including protein quantitation and trypsin digestion. To improve the proteomic compatibility of buffers with high concentration of detergent, we used short gel method to pretreat buffers containing detergent. Protein samples were first separated by a short (2?2.5 mm) SDS-PAGE electrophoresis, and proteins were quantitated by comparing with bovine serum albumin standards via optical density analysis. The gel was then cut and peptides were recovered using in-gel digestion. The quantitative linearity range of this method was 1 to 8 μg. The quantitation was accurate and reproducible. After short gel analysis, recovered peptides generated high mass spectrometry signals. In conclusion, short gel method eliminated the interference of high concentration detergent in the proteomics analysis, and it was suitable for protein samples’ pretreatment, and was worth to apply in proteomic research.

Abstract:In silico acquirement of the accurate residue details of protein on chromatographic media is a bottleneck in protein chromatography separation and purification. Here we developed a novel approach by coupling with H/D exchange and nuclear magnetic resonance to observe hen egg white lysozyme (HEWL) unfolding behavior adsorbed on cation exchange media (SP Sepharose FF). Analysis of 1D 1H NMR shows that protein unfolding accelerated H/D exchange rate, leading to more loss of signal of amide hydrogen owing to exposure of residues and the more unfolding of protein. Analysis of two-dimensional hydrogen-hydrogen total correlation spectroscopy shows that lysozyme lost more signals and experienced great unfolding during its adsorption on media surface. However, for several distinct fragments, the protection degrees varied, the adsorbed lysozyme lost more signal intensity and was less protected at disorder structures (coil, bend, and turn), but was comparatively more protected against exchange at secondary structure domains (α-helix, β-sheet). Finally, the binding site was determined by electrostatic calculations using computer simulation methods in conjunction with hydrogen deuterium labeled protein and NMR. This study would help deeply understand the microscopic mechanism of protein chromatography and guide the purposely design of chromatographic process and media. Moreover, it also provide an effective tool to study the protein and biomaterials interaction in other applications.

Abstract:Three long-chain polyunsaturated fatty acids, docosahexaenoic acid (DHA, 22:6n-3), eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (ARA, 20:4n-6), are the most biologically active polyunsaturated fatty acids in the body. They are important in developing and maintaining the brain function, and in preventing and treating many diseases such as cardiovascular disease, inflammation and cancer. Although mammals can biosynthesize these long-chain polyunsaturated fatty acids, the efficiency is very low and dietary intake is needed to meet the requirement. In this study, a multiple-genes expression vector carrying mammalian △6/△5 fatty acid desaturases and △6/△5 fatty acid elongases coding genes was used to transfect HEK293T cells, then the overexpression of the target genes was detected. GC-MS analysis shows that the biosynthesis efficiency and level of DHA, EPA and ARA were significantly increased in cells transfected with the multiple-genes expression vector. Particularly, DHA level in these cells was 2.5 times higher than in the control cells. This study indicates mammal possess a certain mechanism for suppression of high level of biosynthesis of long chain polyunsaturated fatty acids, and the overexpression of △6/△5 fatty acid desaturases and △6/△5 fatty acid elongases broke this suppression mechanism so that the level of DHA, EPA and ARA was significantly increased. This study also provides a basis for potential applications of this gene construct in transgenic animal to produce high level of these long-chain polyunsaturated fatty acid.

Abstract:Here we discuss whether N terminal sequencing is appropriate as one of the conventional control methods for monoclonal antibody products. We determined the N terminal sequences of two monoclonal antibody products targeting two antigens separately with both Edman degradation and mass peptide spectrometry. We also identified the characteristic peptide fragments with mass spectrometry. Furthermore, we analyzed their heterogeneity with ion exchange chromatography, capillary zone electrophoresis and Imaged Capillary Isoelectric Focusing. Edman degradation method showed that the N terminal 15 amino acids of heavy and light chains of the two monoclonal antibodies were identical. Peptide mass spectrometry demonstrated that T1 peptide fragments of heavy and light chains of the two antibodies were also the same. But in contrast, peptide mapping and the three analytical methods for heterogeneity could effectively identify and differentiate the two antibodies. The N terminal sequences of two monoclonal antibodies are identical because the number of framework sequences of humanized or human monoclonal antibody is relatively limited, so whether N terminal sequencing analysis could be regulated as one of the practical control methods should be carefully discussed. Our work also proves that the above analytical methods could combinatorially applied to the identification of monoclonal antibody products, and are more objective compared to N terminal sequencing.

Abstract:In order to discover the steroid biotransformation ability of ?lamentous fungus Aspergillus niger TCCC41650, we studied the fermentation of 4-androstene-3,17-dione with A. niger TCCC41650. The transformation product was purified, crystallized and determined as 16β-hydroxy-androst-4-ene-3,17-dione by X-ray single crystal diffraction method. The best fermentation condition was found to be pH 6.0, ethanol amount 2% with a substrate concentration of 1‰, the transformation rate is 85.81% after 72 h. Based on the best of our knowledge, 16β-hydroxylation rarely occurs in microbial transformations of steroid. This study laid the foundation for the research of 16β-hydroxylation steroids.

Abstract:Gluconobacter oxydans converts glucose to gluconic acid and subsequently to 5-keto-D-gluconic acid (5-KGA), a precursor of industrially important L(+)-tartaric acid. To increase the yield of 5-KGA, fermentation conditions of 5-KGA production was optimized. Under the optimum medium and culture conditions in the shake flask, the highest 5-KGA production reached 19.7 g/L, increased by 43.8% after optimization. In a 5-L bioreactor, the pH was controlled at 5.5 and dissolved oxygen (DO) at 15%, 5-KGA production reached 46.0 g/L, raised at least 1.3 times than in the shake flask. Glucose feeding fermentation process was further developed, and the highest 5-KGA production of 75.5 g/L with 70% of yield was obtained, 32.0% higher than the highest reported value. Therefore, this newly developed fermentation process provided a practical and effective alternative for the commercial production of 5-KGA, and further of L(+)-tartaric acid.