Endo-xylogalacturonan hydrolase, a novel pectinolytic enzyme

We screened an Aspergillus tubingensis expression library constructed in the yeast Kluyveromyces lactis for xylogalacturonan-hydrolyzing activity in microwell plates by using a bicinchoninic acid assay. This assay detects reducing carbohydrate groups when they are released from a carbohydrate by enzymatic activity. Two K. lactis recombinants exhibiting xylogalacturonan-hydrolyzing activity were found among the 3,400 colonies tested. The cDNA insert of these recombinants encoded a 406-amino-acid protein, designated XghA, which was encoded by a single-copy gene, xghA. A multiple-sequence alignment revealed that XghA was similar to both polygalacturonases (PGs) and rhamnogalacturonases. A detailed examination of conserved regions in the sequences of these enzymes revealed that XghA resembled PGs more. High-performance liquid chromatography and matrix-assisted laser desorption ionization-time of flight mass spectrometry of the products of degradation of xylogalacturonan and saponified modified hairy regions of apple pectin by XghA demonstrated that this enzyme uses an endo type of mechanism. XghA activity appeared to be specific for a xylose-substituted galacturonic acid backbone.     

Expression and characterization of a thermostable β-xylosidase from the hyperthermophile, Thermotoga maritima

A thermostable -xylosidase from a hyperthermophilic bacterium, Thermotoga maritima, was over-expressed in Escherichia coli using the T7 polymerase expression system. The expressed -xylosidase was purified in two steps, heat treatment and immobilized metal affinity chromatography, and gave a single band on SDS-PAGE. The maximum activity on p-nitrophenyl -d-xylopyranoside was at 90 °C and pH 6.1. The purified enzyme had a half-life of over 22-min at 95 °C, and retained over 57% of its activity after holding a pH ranging from 5.4 to 8.5 for 1 h at 80 °C. Among all tested substrates, the purified enzyme had specific activities of 275, 50 and 29 U mg^–1 on pNPX, pNPAF, and pNPG, respectively. The apparent Michaelis constant of the -xylosidase was 0.13 mm for pNPX with a V _max of 280 U mg^–1. When the purified -xylosidase was added to xylanase, corncob xylan was hydrolized completely to xylose.     

Development of an antibiotic-free plasmid selection system based on thymine auxotrophy in Lactococcus lactis

Lactococcus lactis has become the best studied species of the lactic acid bacteria (LAB) clade and an ideal cell factory for heterogenous proteins. We have constructed an antibiotic-free expression vector, pMG-thyA, using thymidine synthase gene thyA as the selection marker. The thyA gene was cloned from the food industry strain Streptococcus thermophilus St-JY and was used to replace the erythromycin resistance genes on L. lactis expression vector pMG36e in order to construct pMG-thyA. The construction of the new vector and thyA-null host L. lactis MG1363-TT yielded an antibiotic-free expression system. The α-amylase gene (amy) was cloned onto the multiple cloning site of the vector pMG-thyA as a reporter gene, yielding the recombinant plasmid pMGthyA-amy. This plasmid was electroporated into L. lactis MG1363-TT, and the recombinant strain grown on SA plates containing 0.5 % (w/v) soluble starch formed distinct bacterial colonies and clear zones (halo) around the colonies following the addition of iodine solution. These research findings lay the foundation for food-grade expression in L. lactis.     

腺苷酸脱氨酶在乳酸克鲁维酵母中构建表达

【目的】实现鼠灰链霉菌来源经密码子优化后的腺苷酸脱氨酶基因在乳酸克鲁维酵母(Kluyveromyces lactis GG799)中组成型表达。【方法】以鼠灰链霉菌(Streptomyces murinus)来源的腺苷酸脱氨酶(AMP)基因经密码子优化后作为模板,设计特异性引物,PCR扩增AMP脱氨酶基因opt-AMPD,以p KLAC1为载体构建重组表达质粒p KLAC1-opt-AMPD,经Sac II线性化后电转化法转入K.lactis GG799,筛选得到重组菌株,测定酶活,经His TrapTM HP纯化后得到AMP脱氨酶,并优化重组菌的发酵培养基。【结果】对AMP脱氨酶基因进行了密码子优化后,构建了重组K.lactis GG799/p KLAC1-opt-AMPD,实现组成型表达,密码子优化后AMP脱氨酶酶活提高到586±50 U/m L。SDS-PAGE结果显示,纯化后的AMP脱氨酶为单一条带,蛋白大小约为60 k D。优化的发酵培养基为(g/L):葡萄糖40、蛋白胨20、酵母粉15、Na Cl 8、KCl 10、Mg SO4 2,30°C、200 r/min发酵120 h,酶活达到2 100±60 U/m L。【结论】实现了密码子优化后的腺苷酸脱氨酶基因在乳酸克鲁维酵母GG799内的组成型表达,为实现腺苷酸脱氨酶的重组高效表达和发酵生产进行了有益探索。     

Cloning and nucleotide sequence of the β-galactosidase gene from Lactococcus lactis ssp. Lactis ATCC7962

The gene encoding -galactosidase of Lactococcus lactis ssp. lactis ATCC7962 was cloned and its nucleotide sequence was determined. The -galactosidase of L. lactis was expressed in Escherichia coli and transformants containing this gene fragment appeared as blue colonies on LB plates containing X-gal. The -galactosidase activity of E. coli transformant was thirty times higher than that of L. lactis. The gene for the 115 kDa -galactosidase has a 2991-bp open reading frame preceded by a putative ribosome binding site. The deduced amino acid sequence show a high degree of homology to the -galactosidase of E. coli, and the putative active site residues are conserved (Glu-429 and Tyr-475)     

Rabies virus glycoprotein expression in Drosophila S2 cells. I: Design of expression/selection vectors, subpopulations selection and influence of sodium butyrate and culture medium on protein expression

The cDNA encoding the rabies virus glycoprotein (RVGP) gene was cloned in expression plasmids under the control of the inductive metallothionein promoter. They were designed in order to bear or not a secretion signal (i) and a cDNA coding for the selection hygromycin. These vectors were transfected into S2 cells, cell populations selected and subpopulations were then obtained by reselection with hygromycin. Cell cultures were examined for kinetics of cell growth, detection of RVGP mRNA and expression of RVGP. All cell populations were shown to express the RVGP mRNA upon induction. S2MtRVGPHy cell population, transfected with one vector that contains RGPV gene and selection gene, was shown to express higher amounts of RVGP as evaluated by flow cytometry (52%) and ELISA (0.64 μg/10⁷ cells at day 7). Subpopulation selection allowed a higher RVGP expression, specially for the S2MtRVGPHy⁺ (5.5 μg/10⁷ cells at day 7). NaBu treatment leading to lower cell growth and higher RVGP expression allowed an even higher RVGP synthesis by S2MtRVGPHy⁺ (8.4 μg/10⁷ cells at day 7). SF900II medium leading to a higher S2MtRVGPHy⁺cell growth allowed a higher final RVGP synthesis in this cell culture. RVGP synthesis may be optimized by the expression/selection vectors design, cell subpopulations selection, chromatin exposure and culture medium employed.     

Susceptibility of influenza A viruses to amantadine is influenced by the gene coding for M protein.

Influenza A virus recombinants derived from "resistant" and "sensitive" parental viruses were examined for susceptibility to inhibition by amantadine. Correlation of gene constellation and amantadine susceptibility revealed that the gene coding for M protein influences sensitivity or resistance to amantadine. All recombinants which derived an M protein from an amantadine-resistant parent were found to be resistant to amantadine. All amantadine-sensitive recombinants derived an M gene from the amantadine-sensitive parent. However, a few amantadine-resistant recombinants which derived an M gene from the sensitive parent were also isolated, suggesting that the expression of amantadine sensitivity in these recombinants may be influenced by other genes.     

乳酸乳球菌AL2基因文库的构建及乳链菌肽生物合成基因簇的筛选

用本实验室获得的乳链菌肽高产菌株乳酸乳球菌AL2总DNA为供体,以λ EMBL3为载体,构建了该菌株的基因文库,共获得3900多个噬斑。通过Southern杂交、PCR扩增及DNA序列测定,证实从该文库中筛选到一个含有完整乳链菌肽生物合成基因簇的重组噬菌体λHJ\|3。     

Effects of diet and exercise on metabolic disturbances in high-fat diet-fed mice

Consumption of a high-fat diet (HFD) is associated with white adipose tissue (WAT) inflammation, which contributes to key components of the metabolic syndrome, including insulin resistance (IR) and hepatic steatosis (HS). To determine the differential effects of exercise training (EX), low-fat diet (LFD), and their combination on WAT inflammation, Balb/cByJ male mice (n = 34) were fed an HFD for 12 wks before they were randomized into one of four intervention groups: HFD-EX, LFD-EX, HFD-sedentary (SED), or LFD-SED. EX mice performed 12 wks of exercise training on a motorized treadmill (1 h/d, 5 d/wk, 12 m/min, 5% grade, 65% VO₂ max), while SED mice remained sedentary in their home cages. WAT gene expression of adipokines was assessed using rt-PCR. IR was measured using HOMA-IR, and HS via hepatic triglyceride content. EX significantly reduced (53%) WAT gene expression of MCP-1, and LFD significantly reduced (50%) WAT gene expression of the macrophage specific marker, F4/80 as well as the adipocytokine IL-1ra (25%). EX independently improved IR, while both EX and LFD improved HS. These findings suggest that both diet and exercise have unique beneficial effects on WAT inflammatory markers and the mechanism by which each treatment improves metabolic complications associated with chronic consumption of an HFD may be different.     

Overexpression of a nitrile hydratase from <Emphasis Type="Italic">Klebsiella oxytoca</Emphasis> KCTC 1686 in <Emphasis Type="Italic">Escherichia coli</Emphasis> and its biochemical characterization

The putative Co-type nitrile hydratase (NHaseK, consisting of α- and β-subunits) genes and the putative activator (17K) gene adjacent to the β subunit region were cloned from Klebsiella oxytoca KCTC 1686. 17K is essential for the functional expression of recombinant NHaseK in Escherichia coli; however, the expression level of 17K was very low when the 17K gene and NHaseK structural genes were expressed as a gene cluster in E. coli BL21(DE3). To improve the 17K expression level and NHaseK activity, the expression cassette was redesigned by placing the 17K gene and NHaseK structural genes under the control of different promoters in the pETDuet-1 expression vector, co-expressing the 17K gene with the gene cluster in a double plasmid or a single plasmid with a double promoter, and introducing an efficient Shine- Dalgarno sequence 5' to the17K gene. The specific activity of NHaseK was improved when 17K was co-expressed with the gene cluster, whereas the production of NHaseK protein decreased. The maximum activity was achieved when an efficient Shine-Dalgarno sequence was introduced 5' to the 17K gene: the expression level of 17K was significantly improved and the expression level of NHaseK did not decrease significantly. The maximum activity was about 63,480 ± 1915.6 U/L broth towards 3-Cyanopyridine. Recombinant NHaseK could hydrolyze a wide range of aliphatic, aromatic, and heterocyclic nitriles, and convert racemic nitriles to the corresponding S-amides, with E values ranging from 9 to 17. The enzyme had a temperature optimum of 35°C and exhibited remarkably stability below 35°C.     

Bed rest increases the amount of mismatched fibers in human skeletal muscle

The effects of a37-day period of bed rest on myosin heavy chain (MHC) expression onboth mRNA and protein level in human skeletal muscle fibers werestudied. Muscle biopsies from vastus lateralis muscle were obtainedfrom seven healthy young male subjects before and after the bed-restperiod. Combined in situ hybridization, immunocytochemistry, and ATPasehistochemistry analysis of serial sections of the muscle biopsiesdemonstrated that fibers showing a mismatch between MHC isoforms at themRNA and protein level increased significantly after the bed-restperiod, suggesting an increase in the amount of muscle fibers in atransitional state. Accordingly, fibers showing a match in expressionof MHC-1 and of MHC-2A at the mRNA and protein level decreased, whereasfibers showing a match between MHC-2X mRNA and protein increased after bed rest. Overall, there was an increase in fibers in a transitional state from phenotypic type 1  2A and 2A  2X.Furthermore, a number of fibers with unusual MHC mRNA and isoproteincombinations were observed after bed rest (e.g., type 1 fibers withonly mRNA for 2X and type 1 fibers negative for mRNA for MHC-/slow,2A, and 2X). In contrast, no changes were revealed after an examination at the protein level alone. These data suggest that the reduced load-bearing activity imposed on the skeletal muscles through bed restwill alter MHC gene expression, resulting in combinations of mRNA andMHC isoforms normally not (or only rarely) observed in musclessubjected to load-bearing activity. On the other hand, the present dataalso show that 37 days of bed rest are not a sufficient stimulus toinduce a similar change at the protein level, as was observed at thegene level.     

Molecular characterization of meiotic recombination within the major histocompatibility complex of the mouse: Mapping of crossover sites within theI region

The molecular analysis of crossing-over within the mouse major histocompatibility complex provides a useful approach for the study of the structural characteristics of meiotic recombination. In this study five intra-I-region recombinants, each derived fromI ^k/I ^b heterozygotes, were characterized for restriction-fragment length polymorphisms (RFLPs) characteristic of theI region of the two parental strains. Southern blot analysis of intra-I recombinant strains A.TBR2, A.TBR3, A.TBR5, A.TBR13, and A.TBR17 using sixI-region DNA probes revealed that the point of crossing-over in all five recombinants occurred within a 6.2-kbKpnI-EcoRI segment located within theE gene. The segments of DNA containing the crossover point from each of the recombinant chromosomes were cloned by screening partial genomic libraries constructed in gt7 bacteriophage. Construction of partial restriction maps of the cloned segments from the parental and recombinant chromosomes permitted the boundaries of the area containing the crossover site to be narrowed to a 4.0-kb segment located almost entirely within an intron of theE gene. The recognition that the points of crossing-over in all five recombinants studied are clustered in a relatively small area of theI region provides further evidence for a hot spot of recombination associated with theE _ß gene.This work was supported by Grants AI14424 and AI20317 from the National Institutes of Health. J. Kobori was supported by a postdoctoral fellowship from the Arthritis Foundation. E. Zimmerer was supported by a postdoctoral fellowship from the Charles and Johanna Busch Fund of the Bureau of Biological Research. D. Spinella was supported by a predoctoral fellowship from the Charles and Johanna Busch Fund.