A lectin gene from the Tiger Milk Mushroom Lignosus rhinocerus TM02® was successfully cloned and expressed via vector pET28a in Escherichia coli BL21(DE3). The recombinant lectin, Rhinocelectin, with a predicted molecular mass of 22.8 kDa, was overexpressed in water-soluble form without signal peptide and purified via native affinity chromatography Ni-NTA agarose. Blast protein analysis indicated the lectin to be homologous to jacalin-related plant lectin. In its native form, Rhinocelectin exists as a homo-tetramer predicted with four chains of identical proteins consisting of 11 beta-sheet structures with only one alpha-helix structure. The antiproliferative activity of the Rhinocelectin against human cancer cell lines was concentration dependent and selective. The IC50 values against triple negative breast cancer cell lines MDA-MB-231 and breast cancer MCF-7 are 36.52 ± 13.55 μg mL-1 and 53.11 ± 22.30 μg mL-1 , respectively. Rhinocelectin is only mildly cytotoxic against the corresponding human nontumorigenic breast cell line 184B5 with IC50 value at 142.19 ± 36.34 μg mL-1 . The IC50 against human lung cancer cell line A549 cells is 46.14 ± 7.42 μg mL-1 while against nontumorigenic lung cell line NL20 is 41.33 ± 7.43 μg mL-1 . The standard anticancer drug, Doxorubicin exhibited IC50 values mostly below 1 μg mL-1 for the cell lines tested. Flow cytometry analysis showed the treated breast cancer cells were arrested at G0/G1 phase and apoptosis induced. Rhinocelectin agglutinated rat and rabbit erythrocytes at a minimal concentration of 3.125 μg mL-1 and 6.250 μg mL-1 , respectively.
Lipolytic enzymes with cold adaptation are gaining increasing interest due to their biotechnological prospective. Previously, a cold adapted family I.3 lipase (AMS8 lipase) was isolated from an Antarctic Pseudomonas. AMS8 lipase was largely expressed in insoluble form. The refolded His-tagged recombinant AMS8 lipase was purified with 23.0% total recovery and purification factor of 9.7. The purified AMS8 lipase migrated as a single band with a molecular weight approximately 65kDa via electrophoresis. AMS8 lipase was highly active at 30°C at pH 10. The half-life of AMS8 lipase was reported at 4 and 2h under the incubation of 30 and 40°C, respectively. The lipase was stable over a broad range of pH. It showed enhancement effect in its relative activity under the presence of Li(+), Na(+), K(+), Rb(+) and Cs(+) after 30min treatment. Heavy metal ions such as Cu(2+), Fe(3+) and Zn(2+) inhibited AMS8 activity. This cold adapted alkalophilic AMS lipase was also active in various organic solvent of different polarity. These unique properties of this biological macromolecule will provide considerable potential for many biotechnological applications and organic synthesis at low temperature.
Beta-glucosidase (BGL) is an important industrial enzyme for food, waste and biofuel processing. Jeotgalibacillus is an understudied halophilic genus, and no beta-glucosidase from this genus has been reported. A novel beta-glucosidase gene (1344 bp) from J. malaysiensis DSM 28777T was cloned and expressed in E. coli. The recombinant protein, referred to as BglD5, consists of a total 447 amino acids. BglD5 purified using a Ni-NTA column has an apparent molecular mass of 52 kDa. It achieved the highest activity at pH 7 and 65 °C. The activity and stability were increased when CaCl2 was supplemented to the enzyme. The enzyme efficiently hydrolyzed salicin and (1 → 4)-beta-glycosidic linkages such as in cellobiose, cellotriose, cellotetraose, cellopentose, and cellohexanose. Similar to many BGLs, BglD5 was not active towards polysaccharides such as Avicel, carboxymethyl cellulose, Sigmacell cellulose 101, alpha-cellulose and xylan. When BglD5 blended with Cellic® Ctec2, the total sugars saccharified from oil palm empty fruit bunches (OPEFB) was enhanced by 4.5%. Based on sequence signatures and tree analyses, BglD5 belongs to the Glycoside Hydrolase family 1. This enzyme is a novel beta-glucosidase attributable to its relatively low sequence similarity with currently known beta-glucosidases, where the closest characterized enzyme is the DT-Bgl from Anoxybacillus sp. DT3-1.
We recently described the production of a detergent-biocompatible crude protease from Streptomyces mutabilis strain TN-X30. Here, we describe the purification, characterization, and immobilization of the serine alkaline protease (named SPSM), as well as the cloning, sequencing, and over-expression of its corresponding gene (spSM). Pure enzyme was obtained after ammonium sulphate precipitation followed by heat-treatment and Sephacryl® S-200 column purification. The sequence of the first 26 NH2-terminal residues of SPSM showed a high sequence identity to subtilisin-like serine proteases produced by actinobacteria. The spSM gene was heterologously expressed in Escherichia coli BL21(DE3)pLysS and E. coli BL21-AI™ strains using pTrc99A (rSPSM) and Gateway™ pDEST™ 17 [(His)6-tagged SPSM] vectors, respectively. Results obtained indicated that the (His)6-tagged SPSM showed the highest stability. The SPSM was immobilized using encapsulation and adsorption-encapsulation approaches and three different carriers. Features of SPSM in soluble and immobilized forms were analyzed by Fourier transform infrared (FTIR) spectroscopy in attenuated total reflection (ATR) mode, X-ray diffraction (XRD), zeta potential measurements, and field emission scanning electron microscopy (FE-SEM). The white clay and kaolin used in this study are eco-friendly binders to alginate-SPSM and show great potential for application of the immobilized SPSM in various industries. Molecular modeling and docking of N-succinyl-l-Phe-l-Ala-l-Ala-l-Phe-p-nitroanilide in the active site of SPSM revealed the involvement of 21 amino acids in substrate binding.
Adiponectin is an adipocyte-secreting hormone that increases cell sensitivity to insulin. It has been previously demonstrated that this hormone protects against Type II Diabetes and, is found to concurrently promote cell proliferation and differentiation. It is postulated that diabetic patients who suffer from tendinopathy may benefit from using adiponectin, which not only improves the metabolism of diabetic ridden tenocytes but also promotes progenitor cell proliferation and differentiation in tendons. These changes may result in tendon regeneration, which, in diabetic tendinopathy, is difficult to treat. Considering that such findings have yet to be demonstrated, a study was thus conducted using diabetic ridden human tenocyte progenitor cells (TPC) exposed to recombinant adiponectin in vitro. TPC were isolated from tendons of diabetic patients and exposed to 10 μg/ml adiponectin. Cell proliferation rate was investigated at various time points whilst qPCR were used to determine the tenogenic differentiation potential. The results showed that adiponectin significantly reduced blood glucose in animal models. The proliferation rate of adiponectin-treated TPCs was significantly higher at 6, 8 and 10 days as compared to untreated cells (p<0.05). The levels of tenogenic genes expression (collagen I, III, tenomodulin and scleraxis) were also significantly upregulated; whilst the osteogenic (Runx2), chondrogenic (Sox9) and adipogenic (PPARУγ) gene expressions remained unaltered. The results of this study suggest that adiponectin is a potential promoter that not only improves diabetic conditions, but also increases tendon progenitor cell proliferation and differentiation. These features supports the notion that adiponectin may be potentially beneficial in treating diabetic tendinopathy.
Klebsiella pneumoniae is a Gram-negative, cylindrical rod shaped opportunistic pathogen that is found in the environment as well as existing as a normal flora in mammalian mucosal surfaces such as the mouth, skin, and intestines. Clinically it is the most important member of the family of Enterobacteriaceae that causes neonatal sepsis and nosocomial infections. In this work, a combination of protein sequence analysis, structural modeling and molecular docking simulation approaches were employed to provide an understanding of the possible functions and characteristics of a hypothetical protein (KPN_02809) from K. pneumoniae MGH 78578. The computational analyses showed that this protein was a metalloprotease with zinc binding motif, HEXXH. To verify this result, a ypfJ gene which encodes for this hypothetical protein was cloned from K. pneumoniae MGH 78578 and the protein was overexpressed in Escherichia coli BL21 (DE3). The purified protein was about 32 kDa and showed maximum protease activity at 30 °C and pH 8.0. The enzyme activity was inhibited by metalloprotease inhibitors such as EDTA, 1,10-phenanthroline and reducing agent, 1,4-dithiothreitol (DTT). Each molecule of KPN_02809 protein was also shown to bind one zinc ion. Hence, for the first time, we experimentally confirmed that KPN_02809 is an active enzyme with zinc metalloprotease activity.
Adiponectin is one of the most bioactive substances secreted by adipose tissue and is involved in the protection against metabolic syndrome, artherosclerosis and type II diabetes. Research into the use of adiponectin as a promising drug for metabolic syndromes requires production of this hormone in high quantities considering its molecular isoforms. The objective of this study is to produce recombinant human adiponectin by Pichia pastoris (P-ADP) as a cheap and convenient eukaryotic expression system for potential application in pharmaceutical therapy. For comparison, adiponectin was also expressed using the Escherichia coli (E-ADP) expression system. Adiponectin was constructed by overlap-extension PCR, and cloned in standard cloning vector and hosts. Recombinant expression vectors were cloned in the P. pastoris and E. coli host strains, respectively. SDS-PAGE and western blotting were used to detect and analyse expressed recombinant protein in both systems. Adiponectin was purified by affinity chromatography and quantified using the Bradford Assay. The results of this study indicated that P-ADP quantity (0.111 mg/mL) was higher than that of E-ADP (0.04 mg/mL) and both were produced in soluble form. However, P-ADP was able to form high molecular weights of adiponectin molecules, whilst E-ADP was not able to form isoforms higher than trimer. In addition, P-ADP was more active in lowering blood glucose compared with E-ADP. The two types of proteins were equally efficient and significantly decreased blood triglyceride and increased high density lipoprotein. We conclude that P. pastoris is able to produce high quantity of bioactive adiponectin for potential use in treatment of metabolic syndromes.
Plastid trnL-trnF and nuclear ribosomal ITS sequences were obtained from selected wild-type individuals of Polygonum minus Huds. in Peninsular Malaysia. The 380 bp trnL-trnF sequences of the Polygonum minus accessions were identical. Therefore, the trnL-trnF failed to distinguish between the Polygonum minus accessions. However, the divergence of ITS sequences (650 bp) among the Polygonum minus accessions was 1%, indicating that these accessions could be distinguished by the ITS sequences. A phylogenetic relationship based on the ITS sequences was inferred using neighbor-joining, maximum parsimony and Bayesian inference. All of the tree topologies indicated that Polygonum minus from Peninsular Malaysia is unique and different from the synonymous Persicaria minor (Huds.) Opiz and Polygonum kawagoeanum Makino.
Klebsiella pneumoniae causes neonatal sepsis and nosocomial infections. One of the strains, K. pneumoniae MGH 78578, shows high level of resistance to multiple microbial agents. In this study, domain family, amino acid sequence and topology analyses were performed on one of its hypothetical protein, YggG (KPN_03358). Structural bioinformatics approaches were used to predict the structure and functionality of YggG protein. The open reading frame (ORF) of yggG, which was a putative metalloprotease gene, was also cloned, expressed and characterized. The ORF was PCR amplified from K. pneumoniae MGH 78578 genomic DNA and cloned into a pET14-b vector for heterologous expression in Escherichia coli. The purified YggG protein was subsequently assayed for casein hydrolysis under different conditions. This protein was classified as peptidase M48 family and subclan gluzincin. It was predicted to contain one transmembrane domain by TMpred. Optimal protein expression was achieved by induction with 0.6 mM isopropyl thiogalactoside (IPTG) at 25 °C for six hours. YggG was purified as soluble protein and confirmed to be proteolytically active under the presence of 1.25 mM zinc acetate and showed optimum activity at 37 °C and pH 7.4. We confirmed for the first time that the yggG gene product is a zinc-dependent metalloprotease.
In recent years, studies on psychrophilic lipases have become an emerging area of research in the field of enzymology. The study described here focuses on the cold-adapted organic solvent tolerant lipase strain Pseudomonas sp. LSK25 isolated from Signy Station, South Orkney Islands, maritime Antarctic. Strain LSK25 lipase was successfully cloned, sequenced, and over-expressed in an Escherichia coli system. Sequence analysis revealed that the lipase gene of Pseudomonas sp. LSK25 consists of 1432 bp, lacks an N-terminal signal peptide and encodes a mature protein consisting of 476 amino acids. The recombinant LSK25 lipase was purified by single-step purification using Ni-Sepharose affinity chromatography and had a molecular mass of approximately 65 kDa. The final recovery and purification fold were 44% and 1.3, respectively. The LSK25 lipase was optimally active at 30 °C and at pH 6. Stable lipolytic activity was reported between temperatures of 5⁻30 °C and at pH 6⁻8. A significant enhancement of lipolytic activity was observed in the presence of Ca2+ ions, the organic lipids of rice bran oil and coconut oil, a synthetic C12 ester and a wide range of water immiscible organic solvents. Overall, lipase strain LSK25 is a potentially desirable candidate for biotechnological application, due to its stability at low temperatures, across a range of pH and in organic solvents.
By a combination of PCR and direct-cycle sequencing using consensus primers, we analyzed approximately 400-bp fragments within the NS3 genes of twenty-one dengue virus type 3 strains isolated from five neighboring Southeast Asian countries at different time intervals from 1956 to 1992. The majority of base disparities were silent mutations, with few predicted amino acid substitutions, thus emphasizing the strict conservation of the NS3 gene. Phylogenetic trees constructed on the basis of these nucleotide differences revealed distinct but related clusters of strains from the Philippines, Indonesia, and strains from Singapore and Malaysia of the 1970s and early 1980s, while the Thai cluster was relatively more distant. This genetic relationship was compatible with that proposed by other workers who have studied other dengue 3 virus genes such as E, M and prM. However, we observed that the more recent, epidemic-associated dengue 3 strains from Singapore and Malaysia of the late 1980s and early 1990s were more closely related to the Thai cluster, implying their evolution from the latter, and emphasizing the importance of viral spread via increasing travel within the Southeast Asian area and elsewhere. Nucleotide sequence analysis of the NS3 genes of dengue viruses can serve to advance the understanding of the epidemiology and evolution of these viruses.
The genes for Nipah virus (NiV) proteins were amplified from viral RNA, cloned into the plasmid pTriEx-3 Hygro, expressed, and purified using immobilized metal affinity chromatography. The recombinant N, F, and G NiV proteins (rNiV-N, rNiV-F, and rNiV-G), were successfully expressed in Escherichia coli and purified with a yield of 4, 16, and 4 mg/L, respectively. All 3 recombinant viral proteins reacted with all 19 samples of NiV-positive human sera. The rNiV-N and rNiV-G proteins were the most immunogenic. The recombinant viral proteins did not react with any of the 12 NiV-negative sera. However, serum from a patient with a late-onset relapsing NiV infection complication was found to be primarily reactive to rNiV-G only. Additionally, there is a distinctive variation in the profile of antigen-reactive bands between the sample from a case of relapsing NiV encephalitis and that of acute NiV infection. The overall findings of this study suggest that the recombinant viral proteins have the potential to be developed further for use in the detection of NiV infection, and continuous biosurveillance of NiV infection in resource-limited settings.
While the capacity for long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis has been elucidated in vertebrates and several invertebrate phyla, the comparative knowledge in crustaceans remains vague. A key obstacle in mapping the full spectrum of LC-PUFA biosynthesis in crustacean is the limited evidence of the functional activities of enzymes involved in desaturation or elongation of polyunsaturated fatty acid substrates. In this present study, we report on the cloning and functional characterization of two Elovl elongases from the orange mud crab, Scylla olivacea. Sequence and phylogenetic analysis suggest these two Elovl as putative Elovl4 and Elovl6, respectively. Using the recombinant expression system in Saccharomyces cerevisiae, we demonstrate the elongation capacity for C18-C22 PUFA substrates in the S. olivacea Elovl4. The S. olivacea Elovl6 elongated saturated fatty acids, monounsaturated fatty acids, and interestingly, C18-C20 PUFA. Taken together, both Elovl fulfill the elongation steps required for conversion of C18 PUFA to their respective LC-PUFA products. Elovl4 is expressed mainly in the hepatopancreas and gill tissues, while Elovl6 is predominant in digestive tissues. The mRNA expression of both enzymes was higher in mud crabs fed with vegetable oil-based diets. Tissue fatty acid composition also showed the existence of LC-PUFA biosynthesis intermediate products in tissues expressing these two elongases. In summary, we report here two novel Elovl with PUFA elongating activities in a marine brachyuran. This will contribute significantly to the understanding of the LC-PUFA biosynthesis pathway in crustaceans and advance the development of aquafeed for intensive farming of the mud crab.
Direct reprogramming of somatic cells into induced pluripotent stem (iPS) cells has emerged as an invaluable method for generating patient-specific stem cells of any lineage without the use of embryonic materials. Following the first reported generation of iPS cells from murine fibroblasts using retroviral transduction of a defined set of transcription factors, various new strategies have been developed to improve and refine the reprogramming technology. Recent developments provide optimism that the generation of safe iPS cells without any genomic modification could be derived in the near future for the use in clinical settings. This review summarizes current and evolving strategies in the generation of iPS cells, including types of somatic cells for reprogramming, variations of reprogramming genes, reprogramming methods, and how the advancement iPS cells technology can lead to the future success of reproductive medicine.
A gene (cbm71) encoding a 71,128-Da mosquitocidal protein (Cbm71) was obtained by screening a size-fractionated XbaI digest of total genomic DNA from Clostridium bifermentans subsp. malaysia CH18 with two gene-specific oligonucleotide probes. The sequence of the Cbm71 protein, as deduced from the sequence of cbm71, corresponds to that of the 66-kDa protein previously described as one of the mosquitocidal components of C. bifermentans subsp. malaysia. Cbm71 shows limited similarities with Bacillus thuringiensis delta-endotoxins, especially in the four first conserved blocks. However, Cbm71 was not immunologically related to any of the Cry toxins and thus belongs to a novel class of mosquitocidal protein. The cbm71 gene was expressed in a nontoxic strain of B. thuringiensis, and Cbm71 was produced during sporulation and secreted to the supernatant of culture. Trichloroacetic-precipitated supernatant preparations were toxic for mosquito larvae of the species Aedes aegypti, Culex pipiens, and Anopheles stephensi.
The non-stereospecific α-haloalkanoic acid dehalogenase DehE from Rhizobium sp. RC1 catalyzes the removal of the halide from α-haloalkanoic acid D,L-stereoisomers and, by doing so, converts them into hydroxyalkanoic acid L,D-stereoisomers, respectively. DehE has been extensively studied to determine its potential to act as a bioremediation agent, but its structure/function relationship has not been characterized. For this study, we explored the functional relevance of several putative active-site amino acids by site-specific mutagenesis. Ten active-site residues were mutated individually, and the dehalogenase activity of each of the 10 resulting mutants in soluble cell lysates against D- and L-2-chloropropionic acid was assessed. Interestingly, the mutants W34→A,F37→A, and S188→A had diminished activity, suggesting that these residues are functionally relevant. Notably, the D189→N mutant had no activity, which strongly implies that it is a catalytically important residue. Given our data, we propose a dehalogenation mechanism for DehE, which is the same as that suggested for other non-stereospecific α-haloalkanoic acid dehalogenases. To the best of our knowledge, this is the first report detailing a functional aspect for DehE, and our results could help pave the way for the bioengineering of haloalkanoic acid dehalogenases with improved catalytic properties.
Psychrophiles are organisms that thrive in cold environments. One of the strategies for their cold adaptation is the ability to synthesize cold-adapted enzymes. These enzymes usually display higher catalytic efficiency and thermolability at lower temperatures compared to their mesophilic and thermophilic counterparts. In this work, a psychrophilic bacterium codenamed pi9 was selected for the cloning of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme in the glycolytic pathway. Here, the cloning of an 1,113 bp fragment of GAPDH gene which covers the 1,002 bp open reading frame by using multiple PCR steps is described. The partial sequence of this gene was PCR amplified by using degenerate primers followed by the cloning of the flanking sequences by inverse and splinkerette PCR techniques. The success in cloning the GAPDH gene by PCR has bypassed the more time consuming genomic library construction and screening method. The full length GAPDH protein was subsequently expressed in E. coli, purified as His-tag protein and confirmed to be catalytically active. This work demonstrated the use of multiple PCR techniques to clone a gene based solely on sequence comparison. It also laid the foundation for further biochemical and structural characterizations of GAPDH from a psychrophilic bacterium by providing a highly purified recombinant protein sample.
Pseudomonas sp. USM 4-55 is a locally isolated bacterium that possesses the ability to produce polyhydroxyalkanoates (PHA) consisting of both poly(3-hydroxybutyrate) [P(3HB)] homopolymer and medium-chain length (mcl) monomers (6 to 14 carbon atoms) when sugars or fatty acids are utilized as the sole carbon source. In this study, the P(3HB) biosynthesis operon carrying the phbC(Ps) P(3HB) synthase was successfully cloned and sequenced using a homologous probe. Three open reading frames encoding NADPH-dependent acetoacetyl-coenzyme A reductase (PhbB(Ps)), beta-ketothiolase (PhbA(Ps)) and P(3HB) synthase (PhbC(Ps)) were found in the phb operon. The genetic organization of phb operon showed a putative promoter region, followed by phbB(Ps)-phbA(Ps)-phbC(Ps). phbR(Ps)which encoded a putative transcriptional activator was located in the opposite orientation, upstream of phbBAC(Ps). Heterologous expression of pGEM''ABex harboring phbC(Ps) in Escherichia coli JM109 resulted in P(3HB) accumulation of up to 40% of dry cell weight (DCW).
The scFv antibody towards the Burkholderia pseudomallei exotoxin was previously constructed by phage display and exhibited good specificity towards the exotoxin. We report here the optimization of the scFv expression in an E. coli expression system. Four different E. coli strains (ER2537, TG1, HB2151, and XL1-Blue) were examined for optimal expression of the scFv protein. Two types of carbon source (i.e. 0.2% glucose and 0.2% glycerol) were also tested for their ability to induce the scFv expression. Cells that carried the scFv construct were grown at 30 degrees C and induced with 0.05 mM IPTG. The expression was then monitored by SDS-PAGE, Western blotting, and indirect ELISA. The Western blot profile showed different levels of the scFv expression among the host strains; XL1-Blue exhibited the highest level of the scFv protein expression. Glycerol at a concentration of 0.2% (v/v) significantly increased the scFv protein expression level when compared to 0.2% (w/v) glucose. Further optimization demonstrated that the scFv protein expression in XL1-Blue was the most optimal with a glycerol concentration as low as 0.05%. However, by indirect ELISA, only the scFv protein that was expressed in 0.2% (v/v) glycerol exhibited high specificity towards the Burkholderia pseudomallei exotoxin.
The xylanase gene from Bacillus pumilus PJ19 amplified by polymerase chain reaction (PCR) was cloned into pCRII vector and transformed into Escherichia coli strain INValphaF'. Starting from an ATG as an initiator codon, an open reading frame coding for 202 amino acids was obtained. The recombinant xylanase sequence showed a 96% homology with the xylanase sequence from B. pumilus IPO strain and had an estimated molecular weight of 22,474. Xylanase activity expressed by E. coli INValphaF' harboring the cloned gene was located primarily in the cytoplasmic fraction.