Enhanced anticancer effect of MAP30-S3 by cyclosproin A through endosomal escape.

Cyclosporin A (CsA) is a calcium antagonist and can enhance the efficacy of some protein drugs, but its mechanism remains unknown. In this study, MAP30, a ribosome-inactivating protein reported to have apoptotic effects on cancer cells, was fused with S3, an epidermal growth factor receptor (EGFR)-targeting peptide. In addition, CsA was used to investigate whether it can further promote the apoptotic effects of S3 fused MAP30 (MAP30-S3). Our result showed that the internalization of FITC-labeled MAP30-S3 was increased significantly by S3 in HeLa cells. Unexpectedly, MAP30-S3 only showed a minor decrease in the viability of EGFR-overexpressing cancer cells, including HeLa, SMMC-7721, and MGC803 (IC50>5 µmol/l). However, 2 µmol/l CsA significantly increased the cytotoxicity of MAP30-S3, especially for HeLa cells (IC50=40.3 nmol/l). In comparison, CsA did not further decrease the cytotoxicity of MAP30-S3 on MRC-5, an EGFR low-expressing cell line from normal lung tissue, indicating that CsA did not affect the cancer-targeting specificity of MAP30-S3. Our results also showed that CsA further increased the apoptotic activity of MAP30-S3 in HeLa cells. CsA could promote the endosomal escape of FITC-MAP30-S3 with a diffused pattern in the cytoplasm. Five endocytic inhibitors were used to investigate the cellular uptake mechanism of MAP30-S3, and the results showed that the endosomal escape-enhancing effect of CsA on MAP30-S3 may be associated with the clathrin-dependent endocytic pathways. Our study suggested that CsA could be a novel endosomal escape enhancer to potentiate the intracellular release of anticancer protein drugs, resulting in their improved therapeutic efficacy.

Increasing thermal stability of glutamate decarboxylase from Escherichia. coli by site-directed saturation mutagenesis and its application in GABA production.

Gamma-amino butyric acid (GABA) is an important bio-product used in pharmaceuticals, functional foods, and a precursor of the biodegradable plastic polyamide 4 (Nylon 4). Glutamate decarboxylase B (GadB) from Escherichia. coli is a highly active biocatalyst that can convert l-glutamate to GABA. However, its practical application is limited by the poor thermostability and only active under acidic conditions of GadB. In this study, we performed site-directed saturation mutagenesis of the N-terminal residues of GadB from Escherichia coli to improve its thermostability. A triple mutant (M6, Gln5Ile/Val6Asp/Thr7Gln) showed higher thermostability, with a 5.6 times (560%) increase in half-life value at 45 °C, 8.7 °C rise in melting temperature (Tm) and a 14.3 °C rise in the temperature at which 50% of the initial activity remained after 15 min incubation (T1550), compared to wild-type enzyme. Protein 3D structure analysis showed that the induced new hydrogen bonds in the same polypeptide chain or between polypeptide chains in E. coli GadB homo-hexamer may be responsible for the improved thermostability. Increased thermostability contributed to increased GABA conversion ability. After 12 h conversion of 3 mol/L l-glutamate, GABA produced and mole conversion rate catalyzed by M6 whole cells was 297 g/L and 95%, respectively, while those by wild-type GAD was 273.5 g/L and 86.2%, respectively.

Improved breast cancer cell-specific intracellular drug delivery and therapeutic efficacy by coupling decoration with cell penetrating peptide and SP90 peptide.

Lack of satisfactory specificity towards tumor cells and poor intracellular delivery efficacy are the major drawbacks with conventional cancer chemotherapy. Conjugated anticancer drugs to targeting moieties e.g. to peptides with the ability to recognize cancer cells and to cell penetrating peptide can improve these characteristics, respectively. Combining a tumor homing peptide with an appropriate cell-penetrating peptide can enhance the tumor-selective internalization efficacy of the carrying cargo molecules. In the present study, the breast cancer homing ability of SP90 peptide and the synergistic effect of SP90 with a cell-penetrating peptide(C peptide) were evaluated. SP90 and chimeric peptide SP90-C specifically targeted cargo molecule into breast cancer cells, especially triple negative MDA-MB-231 cell, in a dose- and time-dependent manner, but not normal breast cells and other cancer cells, while C peptide alone had no cell-selectivity. SP90-C increased the intracellular delivery efficiency by 12-fold or 10-fold compared to SP90 or C peptide alone, respectively. SP90 and SP90-C conjugation increased the anti-proliferative and apoptosis-inducing activity of HIV-1 Vpr, a potential novel anticancer protein drug, to breast cancer cell but not normal breast cell by arresting cells in G2/M phase. With excellent breast cancer cell-selective penetrating efficacy, SP90-C appears as a promising candidate vector for targeted anti-cancer drug delivery. SP90-VPR-C is a potential novel breast cancer-targeted anticancer agent for its high anti-tumor activity and low toxicity.

Heterologous overexpression of Vigna radiata epoxide hydrolase in Escherichia coli and its catalytic performance in enantioconvergent hydrolysis of p-nitrostyrene oxide into (R)-p-nitrophenyl glycol.

Two native epoxide hydrolases (EHs) were previously discovered from mung bean powder (Vigna radiata), both of which can catalyze the enantioconvergent hydrolysis of p-nitrostyrene oxide (pNSO). In this study, the encoding gene of VrEH1 was successfully cloned from the cDNA of V. radiata by RT-PCR and rapid amplification of cDNA ends (RACE) technologies. High homologies were found to two putative EHs originated from Glycine max (80%) and Medicago truncatula (79%). The vreh1 gene constructed in pET28a(+) vector was then heterologously overexpressed in Escherichia coli BL21(DE3), and the encoded protein was purified to homogeneity by nickel affinity chromatography. It was shown that VrEH1 has an optimum activity at 45 °C and is very thermostable with an inactivation energy of 468 kJ mol(-1). The enzyme has no apparent requirement of metal ions for activity, and its activity was strongly inhibited by 1 mM of Ni(2+), Cu(2+), Fe(2+), or Co(2+). By adding 0.1% Triton X-100, the enzyme activity could be significantly increased up to 340%. VrEH1 shows an unusual ability of enantioconvergent catalysis for the hydrolysis of racemic pNSO, affording (R)-p-nitrophenyl glycol (pNPG). It displays opposite regioselectivity toward (S)-pNSO (83% to Cα) in contrast to (R)-pNSO (87% to Cß). The K M and k cat of VrEH1 were determined to be 1.4 mM and 0.42 s(-1) for (R)-pNSO and 5.5 mM and 6.2 s(-1) for (S)-pNSO. This thermostable recombinant VrEH1 with enantioconvergency is considered to be a promising biocatalyst for the highly productive preparation of enantiopure vicinal diols and also a good model for understanding the mechanism of EH stereoselectivity.

Improvement of ethanol productivity and energy efficiency by degradation of inhibitors using recombinant Zymomonas mobilis (pHW20a-fdh).

Toxic compounds, such as formic acid, furfural, and hydroxymethylfurfural (HMF) generated during pretreatment of corn stover (CS) at high temperature and low pH, inhibit growth of Zymomonas mobilis and lower the conversion efficiency of CS to biofuel and other products. The inhibition of toxic compounds is considered as one of the major technical barriers in the lignocellulose bioconversion. In order to detoxify and/or degrade these toxic compounds by the model ethanologenic strain Z. mobilis itself in situ the fermentation medium, we constructed a recombinant Z. mobilis ZM4 (pHW20a-fdh) strain that is capable of degrading toxic inhibitor, formate. This is accomplished by cloning heterologous formate dehydrogenase gene (fdh) from Saccharomyces cerevisiae and by coupling this reaction of NADH regeneration reaction system with furfural and HMF degradation in the recombinant Z. mobilis strain. The NADH regeneration reaction also improved both the energy efficiency and cell physiological activity of the recombinant organism, which were definitely confirmed by the improved cell growth, ethanol yield, and ethanol productivity during fermentation with CS hydrolysate.

Expression and characterization of a novel enantioselective lipase from Aspergillus fumigatus.

A 1,080-bp cDNA (CGMCC 2873) encoding of a cold-active lipase of Aspergillus fumigatus (AFL67) was cloned and expressed in Escherichia coli for the first time. The new lipase, AFL67, was one-step purified by 8.30 folds through Ni-NTA affinity chromatography with a recovery of 86.8 %. The specific activity of purified AFL67 was 449 U mg(-1) on p-NP hexanoate. AFL67 preferentially hydrolyzed p-nitrophenyl esters of short- and medium-chain fatty acids, with p-nitrophenyl hexanoate the maximum. The optimum temperature and pH was 15 °C and 7.5, respectively. The purified AFL67 was stable at 10-25 °C for 30 min, and in the pH range of 6.0-9.0 for 16 h (at 4 °C). Its activity was increased by 47 and 50 %, in the presence of 10 % (v/v) ethanol and isopropanol, respectively. The new lipase AFL67 highly enantioselectively deacylated (S)-α-acetoxyphenylacetic acid (APA) and o-Cl-APA, m-Cl-APA, and p-Cl-APA to (S)-mandelic acid and its derivates. These features render this cold-active novel lipase AFL67 attractive for biotechnological applications in the field of enantioselective synthesis of chiral mandelic acids, o-acylated mandelic acids, and their derivates and detergent additives.

Expression and characterization of a novel lipase from Aspergillus fumigatus with high specific activity.

A novel lipase gene from Aspergillus fumigatus, afl1-1, was cloned and expressed with a molecular mass of 38 kDa in Escherichia coli for the first time. The recombinant lipase had a preference for short carbon chain p-nitrophenyl esters, especially toward C2 p-nitrophenyl ester and exhibited potent hydrolysis activity that had not been observed. The optimum pH and temperature of this new enzyme were 8.5 and 65 °C, respectively. The recombinant lipase (AFL1-1) is an alkaline enzyme which was stable in the pH range 6.0∼8.5 for 16 h (at 4 °C) and at 30∼50 °C for 1 h. It is an intracellular enzyme which was purified approximately 8.47-fold with an overall yield of 86.1% by single-step Ni-NTA affinity purification, with a very high specific activity of approximately 1.00 × 10(3) U mg(-1) on a standard substrate of p-nitrophenyl acetate. The Michaelis-Menten kinetic parameters V (max) and K (m) of the lipase were 1.37 mM mg(-1) min(-1) and 14.0 mM, respectively. Ca(2+) and other metal ions could not activate the lipase. According to the homology analysis and site-directed mutagenesis assay, the catalytic triad of the recombinant lipase was identified as Ser-165, Asp-260, and His-290 residues.

Gene cloning and expression of a novel hypoglycaemic peptide from Momordica charantia.

BACKGROUND: Momordica charantia (MC) is used in many Asian countries as a traditional functional food and medicine. Polypeptide-P, a 166 amino acid (AA) polypeptide isolated from MC seeds, has been reported to show hypoglycaemic effects in patients with type I or type II diabetes. The AA sequence of this peptide has been determined, but its gene sequence has yet to be published. RESULTS: In this study a gene-cloning strategy was employed to obtain the polypeptide-P gene sequence using degenerate reverse transcription polymer chain reaction and genome-walking methods. A complete 498 bp sequence encoding the polypeptide-P protein was cloned from MC seeds. Subsequent assays of the bioactivity of the expressed recombinant protein revealed that it had significant hypoglycaemic activity in alloxan-induced diabetic mice. This result suggests that recombinant polypeptide-P has hypoglycaemic effects. CONCLUSION: This is the first report of cloning and expression of the MC polypeptide-P gene. The cloned gene could be helpful for exploring the mechanisms of polypeptide-P gene expression and regulation in MC. Furthermore, this gene could be used as a potential tool both for screening MC varieties with high hypoglycaemically active substance content and for breeding new varieties of MC with high economic value, which could in turn be beneficial to farmers.

A novel human derived cell-penetrating peptide in drug delivery.

Cell-penetrating peptides can carry a variety of biologically active molecules into cells. Here we have identified a novel CPP derived from the C-terminus of human extracellular superoxide dismutase (hC-SOD3) which was shown to be located throughout in the cytoplasm and nucleus by fluorescence microscopy investigation. Furthermore, when apoptin fused to hC-SOD3, it was translocated efficiently into HeLa cells resulting in antitumor activities. This study shows that hC-SOD3 has the potential to penetrate and translocate cargo molecules into cells and has no cytotoxicity at effective concentration.

Significantly improved expression and biochemical properties of recombinant Serratia marcescens lipase as robust biocatalyst for kinetic resolution of chiral ester.

A lipase gene from Serratia marcescens ECU1010 was cloned into expression vector pET28a, sequenced, and overexpressed as an N terminus His-tag fusion protein in Escherichia coli. Through the optimization of culture conditions in shake flask, the lipase activity was improved up to 1.09 x 105 U/l, which is a great improvement compared to our previous reports. It was purified to homogeneity by Ni-NTA affinity chromatography with an overall yield of 59.4% and a purification factor of 2.4-fold. This recombinant lipase displayed excellent stability below 30 °C and within the pH range of 5.0-6.8, giving temperature and pH optima at 40 °C and pH 9.0, respectively. The lipase activity was found to increase in the presence of metal ions such as Ca²+, Cu²+, and some nonionic surfactants such as PEG series. In addition, among p-nitrophenyl esters of fatty acids with varied chain length, the recombinant lipase showed the maximum activity on p-nitrophenyl laurate (C12). Using racemic trans-3-(4'-methoxy-phenyl)-glycidyl methyl ester [(±)-MPGM] as substrate, which is a key chiral synthon for production of diltiazem, a 50% conversion yield was achieved after 4 h in toluene-water (100 mM KPB phosphate buffer, pH 7.5) biphasic system (5:5 ml) at 30 °C under shaking condition (160 rpm), affording (-)-MPGM in nearly 100% ee. The K(m) and V(max) values of the lipase for (±)-MPGM were 222 mM and 1.24 mmol min⁻¹ mg⁻¹, respectively. The above-mentioned features make the highly enantioselective lipase from Serratia marcescens ECU1010 a robust biocatalyst for practical use in large-scale production of diltiazem intermediate.

Identification and characterization of an androgen-responsive Kap promoter enhancer located in the intron II region of human angiotensinogen gene.

Transgenic expression of the human angiotensinogen (HAGT) gene directed by the mouse kidney androgen-regulated protein (Kap) gene promoter is proximal tubule cell-specific and androgen-regulated in vivo. The same Kap promoter fragment did not support similar regulation of other genes, but a transgene based on the original chimeric KAP-hAGT construct successfully directed NHE3 to kidney, suggesting that sequences within the HAGT gene fragment of the construct contributed to the regulation of its expression in vivo. In the present study, androgen-responsive regulatory sequences in the HAGT gene portions of the transgene were examined in transfected renal cells. A 1.4-kb enhancer between exons 2 and 3 was identified that increased the basal expression of Kap promoter 1.5- to 2-fold, its induction by dihydrotestosterone (DHT) 2- to 3-fold and its induction by dexamethasone (Dex) 4- to 5-fold. Sequence analysis revealed two potential hormone-responsive elements. Mutational assays and electrophoretic mobility shift assay showed one of these elements was androgen-specific. These findings may influence future strategies for the design of inducible, cell-specific transgenes.

Biocatalytic properties of a recombinant Fusarium proliferatum lactonase with significantly enhanced production by optimal expression in Escherichia coli.

The levo-lactonase gene of Fusarium proliferatum ECU2002 (EC3.1.1.25) was cloned and expressed in Escherichia coli JM109 (DE3) for biocatalytic resolution of industrially important chiral lactones, including DL-pantoyl lactone which was a key precursor to calcium D-pantothenate. By increasing the biomass concentration and lowering the inducer (isopropyl-beta-D-thiogalactoside) concentration and induction temperature, the lactonase production was significantly enhanced up to 20 kU/L, which was 20 times higher than that of wild-type strain F. proliferatum ECU2002. The recombinant Fusarium lactonase was purified using immobilized metal affinity chromatography, and its SDS-PAGE revealed a molecular mass of 50 kDa for the recombinant protein, suggesting that the enzyme was a simplex protein. Furthermore, biocatalytic properties of the recombinant lactonase were investigated, including kinetic parameters, additive's effect, and substrate specificity. The results reported in this paper provide a feasible method to make the whole cells of E. coli JM109 (DE3) expressing lactonase gene to be a highly efficient and easy-to-make biocatalyst for asymmetric synthesis of chiral compounds.

[Antioxidant activity of natural and cultured Cordyceps sp].

OBJECTIVE: Investigating the antioxidant activities of water and ethanol extracts of natural Cordyceps sinensis and Cordyceps militaris and their fermentation preparations. METHOD: The samples were tested through 6 assays: inhibition ability of linoleic acid oxidation; scavenging activity of DPPH, hydrogen peroxide, hydroxyl radical and superoxide anion; and metal chelating activity. RESULT: Samples showed different antioxidant ability, and there was not an extract that exhibited high activity in all assays; however, water extract of natural C. militaris could be regarded as the most powerful antioxidant among 8 samples. It had high activity in inhibition of linoleic acid oxidation, chelating metal ions, and scavenging DPPH and hydroxyl radical. The research also indicated that the contents of phenolic compounds in water and ethanol extracts of natural and cultured Cordyceps sp. had huge difference. CONCLUSION: Natural Cordyceps sp. and its fermentation preparations could be used as potential natural antioxidants. The fermented process affected the antioxidant ability of cultured Cordyceps sp., and the antioxidant activity of both natural and cultured Cordyceps sp. did not significantly related with the quantity of phenolics.

[Coexpression of caiB and caiE with two plasmids in E. coli].

Recombinant bacteria exhibiting high enzymatic activities were obtained by cloning and coexpression of both caiB gene and caiE gene in the host of E. coli Bl21(DE3), which encode carnitine dehydratase and a protein related to the synthesis of cofactor for carnitine dehydratase, respectively. In order to coexpress these two genes, compatible and incompatible two plasmids system were used, the difference between them was also studied. After induction with IPTG, both caiB and caiE genes were coexpressed in both compatible two plasmids system and incompatible two plasmids system. In the former system, the expressed products accounted for 17% and 10% of the total proteins in the host; in the later system, the proportion was 39% and 20%, respectively. The activity of carnitine dehydratase in E. coli Bl21(DE3) with either coexpression system is about 2.3 times than that in E. coli Bl21(DE3) with only pET28-caiB. The plasmids stabilities in these two systems were the same, all needed the help of antibiotic selective pressure.

Cloning, Sequencing and Expression of Human Copper, Zinc-superoxide Dismutase cDNA.

The cDNA encoding the human copper, zinc-superoxide dismutase(Cu, Zn-SOD) was amplified from the human liver by RT-PCR and sequenced. The cloned human Cu, Zn-SOD cDNA was ligated into expression vector pET-22b(+) under T7 promotor. After 3 h induction with 1 mmol/L IPTG, human Cu, Zn-SOD was highly expressed in E.coli BL21(DE3). The expression product was up to 30% of the total protein of the bacteria in soluble form, which had specific SOD activity.