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Rapid cloning of disease-resistance genes in plants using mutagenesis and sequence capture

Steuernagel B, Periyannan SK, Hernández-Pinzón I, Witek K, Rouse MN, Yu G, et al.

Nat Biotechnol, 2016 Jun;34(6):652-5.
PMID: 27111722 DOI: 10.1038/nbt.3543

Wild relatives of domesticated crop species harbor multiple, diverse, disease resistance (R) genes that could be used to engineer sustainable disease control. However, breeding R genes into crop lines often requires long breeding timelines of 5-15 years to break linkage between R genes and deleterious alleles (linkage drag). Further, when R genes are bred one at a time into crop lines, the protection that they confer is often overcome within a few seasons by pathogen evolution. If several cloned R genes were available, it would be possible to pyramid R genes in a crop, which might provide more durable resistance. We describe a three-step method (MutRenSeq)-that combines chemical mutagenesis with exome capture and sequencing for rapid R gene cloning. We applied MutRenSeq to clone stem rust resistance genes Sr22 and Sr45 from hexaploid bread wheat. MutRenSeq can be applied to other commercially relevant crops and their relatives, including, for example, pea, bean, barley, oat, rye, rice and maize.

Matched MeSH terms: Cloning, Molecular/methods*
Fulltext Isolation and characterization of CCoAOMT in interspecific hybrid of Acacia auriculiformis x Acacia mangium--a key gene in lignin biosynthesis

Pang SL, Ong SS, Lee HH, Zamri Z, Kandasamy KI, Choong CY, et al.

Genet. Mol. Res., 2014;13(3):7217-38.
PMID: 25222227 DOI: 10.4238/2014.September.5.7

This study was directed at the understanding of the function of CCoAOMT isolated from Acacia auriculiformis x Acacia mangium. Full length cDNA of the Acacia hybrid CCoAOMT (AhCCoAOMT) was 1024-bp long, containing 750-bp coding regions, with one major open reading frame of 249 amino acids. On the other hand, full length genomic sequence of the CCoAOMT (AhgflCCoAOMT) was 2548 bp long, containing three introns and four exons with a 5' untranslated region (5'UTR) of 391 bp in length. The 5'UTR of the characterized CCoAOMT gene contains various regulatory elements. Southern analysis revealed that the Acacia hybrid has more than three copies of the CCoAOMT gene. Real-time PCR showed that this gene was expressed in root, inner bark, leaf, flower and seed pod of the Acacia hybrid. Downregulation of the homologous CCoAOMT gene in tobacco by antisense (AS) and intron-containing hairpin (IHP) constructs containing partial AhCCoAOMT led to reduction in lignin content. Expression of the CCoAOMT in AS line (pART-HAS78-03) and IHP line (pART-HIHP78-06) was reduced respectively by 37 and 75% compared to the control, resulting in a decrease in the estimated lignin content by 24 and 56%, respectively. AhCCoAOMT was found to have altered not only S and G units but also total lignin content, which is of economic value to the pulp industry. Subsequent polymorphism analysis of this gene across eight different genetic backgrounds each of A. mangium and A. auriculiformis revealed 47 single nucleotide polymorphisms (SNPs) in A. auriculiformis CCoAOMT and 30 SNPs in A. mangium CCoAOMT.

Matched MeSH terms: Cloning, Molecular
Fulltext Functional expression of a novel α-amylase from Antarctic psychrotolerant fungus for baking industry and its magnetic immobilization

He L, Mao Y, Zhang L, Wang H, Alias SA, Gao B, et al.

BMC Biotechnol, 2017 02 28;17(1):22.
PMID: 28245836 DOI: 10.1186/s12896-017-0343-8

BACKGROUND: α-Amylase plays a pivotal role in a broad range of industrial processes. To meet increasing demands of biocatalytic tasks, considerable efforts have been made to isolate enzymes produced by extremophiles. However, the relevant data of α-amylases from cold-adapted fungi are still insufficient. In addition, bread quality presents a particular interest due to its high consummation. Thus developing amylases to improve textural properties could combine health benefits with good sensory properties. Furthermore, iron oxide nanoparticles provide an economical and convenient method for separation of biomacromolecules. In order to maximize the catalytic efficiency of α-amylase and support further applications, a comprehensive characterization of magnetic immobilization of α-amylase is crucial and needed.
RESULTS: A novel α-amylase (AmyA1) containing an open reading frame of 1482 bp was cloned from Antarctic psychrotolerant fungus G. pannorum and then expressed in the newly constructed Aspergillus oryzae system. The purified recombinant AmyA1 was approximate 52 kDa. AmyA1 was optimally active at pH 5.0 and 40 °C, and retained over 20% of maximal activity at 0-20 °C. The K m and V max values toward soluble starch were 2.51 mg/mL and 8.24 × 10-2 mg/(mL min) respectively, with specific activity of 12.8 × 103 U/mg. AmyA1 presented broad substrate specificity, and the main hydrolysis products were glucose, maltose, and maltotetraose. The influence of AmyA1 on the quality of bread was further investigated. The application study shows a 26% increase in specific volume, 14.5% increase in cohesiveness and 14.1% decrease in gumminess in comparison with the control. AmyA1 was immobilized on magnetic nanoparticles and characterized. The immobilized enzyme showed improved thermostability and enhanced pH tolerance under neutral conditions. Also, magnetically immobilized AmyA1 can be easily recovered and reused for maximum utilization.
CONCLUSIONS: A novel α-amylase (AmyA1) from Antarctic psychrotolerant fungus was cloned, heterologous expression in Aspergillus oryzae, and characterized. The detailed report of the enzymatic properties of AmyA1 gives new insights into fungal cold-adapted amylase. Application study showed potential value of AmyA1 in the food and starch fields. In addition, AmyA1 was immobilized on magnetic nanoparticles and characterized. The improved stability and longer service life of AmyA1 could potentially benefit industrial applications.

Matched MeSH terms: Cloning, Molecular
Fulltext Sequence analysis of the PIP5K locus in Eimeria maxima provides further evidence for eimerian genome plasticity and segmental organization

Song BK, Pan MZ, Lau YL, Wan KL

Genet. Mol. Res., 2014;13(3):5803-14.
PMID: 25117339 DOI: 10.4238/2014.July.29.8

Commercial flocks infected by Eimeria species parasites, including Eimeria maxima, have an increased risk of developing clinical or subclinical coccidiosis; an intestinal enteritis associated with increased mortality rates in poultry. Currently, infection control is largely based on chemotherapy or live vaccines; however, drug resistance is common and vaccines are relatively expensive. The development of new cost-effective intervention measures will benefit from unraveling the complex genetic mechanisms that underlie host-parasite interactions, including the identification and characterization of genes encoding proteins such as phosphatidylinositol 4-phosphate 5-kinase (PIP5K). We previously identified a PIP5K coding sequence within the E. maxima genome. In this study, we analyzed two bacterial artificial chromosome clones presenting a ~145-kb E. maxima (Weybridge strain) genomic region spanning the PIP5K gene locus. Sequence analysis revealed that ~95% of the simple sequence repeats detected were located within regions comparable to the previously described feature-rich segments of the Eimeria tenella genome. Comparative sequence analysis with the orthologous E. maxima (Houghton strain) region revealed a moderate level of conserved synteny. Unique segmental organizations and telomere-like repeats were also observed in both genomes. A number of incomplete transposable elements were detected and further scrutiny of these elements in both orthologous segments revealed interesting nesting events, which may play a role in facilitating genome plasticity in E. maxima. The current analysis provides more detailed information about the genome organization of E. maxima and may help to reveal genotypic differences that are important for expression of traits related to pathogenicity and virulence.

Matched MeSH terms: Cloning, Molecular
Molecular characterization and expression of a putative ATPase domain from Eimeria tenella

Chong SP, Jangi MS, Wan KL

J. Biochem. Mol. Biol. Biophys., 2002 Apr;6(2):123-8.
PMID: 12186768

VCP (Valosin-Containing Protein), a member of the AAA (ATPases Associated to a variety of cellular Activities) family of proteins, possesses a duplicated highly conserved ATPase domain. An expressed sequence tag (EST), representing a clone from the Eimeria tenella merozoite cDNA library, was found to have high similarity to VCP genes from other organisms. A complete sequence derived from the corresponding clone (designated eth060) shows amino acid identity of 42-62% with other members of the VCP subfamily. Sequence analysis identified a putative ATPase domain in the eth060 sequence. This domain was PCR-amplified using gene-specific primers and cloned into a pBAD/Thio-TOPO expression vector. Expression in Escherichia coli demonstrated that the putative ATPase domain, which consists of 414 amino acid residues, produced a fusion protein of approximately 60 kDa in size.

Matched MeSH terms: Cloning, Molecular
Investigation of highly unsaturated fatty acid metabolism in the Asian sea bass, Lates calcarifer

Mohd-Yusof NY, Monroig O, Mohd-Adnan A, Wan KL, Tocher DR

Fish Physiol Biochem, 2010 Dec;36(4):827-43.
PMID: 20532815 DOI: 10.1007/s10695-010-9409-4

Lates calcarifer, commonly known as the Asian sea bass or barramundi, is an interesting species that has great aquaculture potential in Asia including Malaysia and also Australia. We have investigated essential fatty acid metabolism in this species, focusing on the endogenous highly unsaturated fatty acid (HUFA) synthesis pathway using both biochemical and molecular biological approaches. Fatty acyl desaturase (Fad) and elongase (Elovl) cDNAs were cloned and functional characterization identified them as ∆6 Fad and Elovl5 elongase enzymes, respectively. The ∆6 Fad was equally active toward 18:3n-3 and 18:2n-6, and Elovl5 exhibited elongation activity for C18-20 and C20-22 elongation and a trace of C22-24 activity. The tissue profile of gene expression for ∆6 fad and elovl5 genes, showed brain to have the highest expression of both genes compared to all other tissues. The results of tissue fatty acid analysis showed that the brain contained more docosahexaenoic acid (DHA, 22:6n-3) than flesh, liver and intestine. The HUFA synthesis activity in isolated hepatocytes and enterocytes using [1-(14)C]18:3n-3 as substrate was very low with the only desaturated product detected being 18:4n-3. These findings indicate that L. calcarifer display an essential fatty acid pattern similar to other marine fish in that they appear unable to synthesize HUFA from C18 substrates. High expression of ∆6 fad and elovl5 genes in brain may indicate a role for these enzymes in maintaining high DHA levels in neural tissues through conversion of 20:5n-3.

Matched MeSH terms: Cloning, Molecular
Isolation and characterization of an oil palm constitutive promoter derived from a translationally control tumor protein (TCTP) gene

Masura SS, Parveez GK, Ti LL

Plant Physiol Biochem, 2011 Jul;49(7):701-8.
PMID: 21549610 DOI: 10.1016/j.plaphy.2011.04.003

We have characterized an oil palm (Elaeis guineensis Jacq.) constitutive promoter that is derived from a translationally control tumor protein (TCTP) gene. The TCTP promoter was fused transcriptionally with the gusA reporter gene and transferred to monocot and dicot systems in order to study its regulatory role in a transient expression study. It was found that the 5' region of TCTP was capable of driving the gusA expression in all the oil palm tissues tested, including immature embryo, embryogenic callus, embryoid, young leaflet from mature palm, green leaf, mesocarp and stem. It could also be used in dicot systems as it was also capable of driving gusA expression in tobacco leaves. The results indicate that the TCTP promoter could be used for the production of recombinant proteins that require constitutive expression in the plant system.

Matched MeSH terms: Cloning, Molecular
Cloning, expression, and purification of recombinant protein from a single synthetic multivalent construct of Mycobacterium tuberculosis

Fang CM, Zainuddin ZF, Musa M, Thong KL

Protein Expr Purif, 2006 Jun;47(2):341-7.
PMID: 16510294 DOI: 10.1016/j.pep.2005.12.007

Tuberculosis remains a major infectious disease with over 8 million new cases and 2 million deaths annually. Therefore, a vaccine more potent than BCG is desperately needed. In this regard, an approximately 800 bp DNA encoding a mycobacterial synthetic gene designated as VacIII (containing ubiquitin gene UbGR and four immunogenic mycobacterial epitopes or genes of ESAT-6, Phos1, Hsp 16.3, and Mtb8.4) was sub-cloned into a bacterial expression vector of pRSET-B resulting in a 6 x His-VacIII fusion gene construction. This recombinant clone was over expressed in Escherichia coli BL-21 (DE-3). The expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in cell lysate. The inclusion bodies were solubilized with 8M urea and the recombinant protein was purified by Ni-NTA column and dialyzed by urea gradient dialysis. This method produced a relatively high yield of recombinant VacIII protein and the cloned VacIII gene offers the potential development of other vaccine formats such as DNA vaccine and recombinant vaccine.

Matched MeSH terms: Cloning, Molecular
Molecular characterisation of six alternatively spliced variants and a novel promoter in human peroxisome proliferator-activated receptor alpha

Chew CH, Samian MR, Najimudin N, Tengku-Muhammad TS

Biochem Biophys Res Commun, 2003 May 30;305(2):235-43.
PMID: 12745064

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a ligand-activated transcriptional factor that governs many biological processes, including lipid metabolism, inflammation, and atherosclerosis. We demonstrate here the existence of six variants and multiple transcriptional start sites of the 5(') untranslated region (UTR) of hPPARalpha gene, originating from the use of alternative splicing mechanisms and four different promoters. Three new novel exons at the 5(')-untranslated region of human PPARalpha gene were also identified and designated as Exon A, Exon B, and Exon 2b. In addition, 1.2kb promoter fragment which drives the transcription of 2 variants with Exon B (hPPARalpha4 and 6) was successfully cloned and characterised. Sequencing results revealed promoter B did not contain a conservative TATA box within the first 100 nucleotides from transcriptional start site but has several GC-rich regions and putative Sp1 sites. Using luciferase reporter constructs transfected into HepG2 and Hep3B cell lines, promoter B was shown to be functionally active. Basal transcriptional activity was significantly high in the promoter fragment -341/+34, but lower in the region -341/-1147 as compared to the fragment -341/+34, indicating the presence of an element conferring transcriptional activation between positions -341 and +34 or alternatively, the presence of transcriptional repression between positions -341 and -1147 in the promoter B of hPPARalpha.

Matched MeSH terms: Cloning, Molecular
Molecular cloning and characterisation of peroxisome proliferator activated receptor gamma1 (PPARgamma1) cDNA gene from guinea pig (Cavia porcellus): tissue distribution

Khoo BY, Samian MR, Najimudin N, Tengku Muhammad TS

Comp Biochem Physiol B Biochem Mol Biol, 2003 Jan;134(1):37-44.
PMID: 12524031

The coding region of guinea pig peroxisome proliferator activated receptor gamma1 (gpPPARgamma1) cDNA was successfully cloned from adipose tissue by reverse transcription polymerase chain reaction (RT-PCR) using the designated primers based on the conserved regions of the other mammalian PPARgamma1 sequence. From RT-PCR, a combination of three cDNA fragments that comprised of the full length coding region PPARgamma1 cDNA gene were amplified, with the size of 498, 550 and 557 bp, respectively. All three fragments were then successfully assembled by utilising the internal restriction sites present at the overlapping regions to give rise to the full-length coding region of gpPPARgamma1 with the size of 1428 bp and consisting of 475 amino acids. Guinea pig PPARgamma1 is highly conserved with those of other species at protein and nucleotide levels. Gene expression studies showed that gpPPARgamma mRNA was predominantly expressed in adipose tissue followed by lung and spleen. However, at the protein level, PPARgamma was also found to be expressed in skeletal muscle.

Matched MeSH terms: Cloning, Molecular
Cloning, expression and display of the PreS domain of hepatitis B virus on filamentous bacteriophage M13

Kok WL, Yusoff K, Nathan S, Tan WS

J. Biochem. Mol. Biol. Biophys., 2002 Feb;6(1):55-8.
PMID: 12186783

The PreS domain of hepatitis B virus (HBV) is believed to be involved in virion assembly and attachment to a hepatocyte receptor during infection. In order to study the functions of this region, we fused it to the g3p protein of bacteriophage M13 that allows the fusion protein to be displayed at the tip of the filament. The fusion protein was detected by the anti-E tag antibody on a Western blot. The polypeptide in a soluble form was produced by transfecting a non-suppressor E. coli host cell with the recombinant phagemid. The soluble protein was detected in cytoplasm, in the periplasmic space and also in the medium. The functional display of the PreS domain would provide an alternative means to study its interactions with the nuleocapsid and hepatocytes.

Matched MeSH terms: Cloning, Molecular
Virus-like particle of Macrobrachium rosenbergii nodavirus produced in Spodoptera frugiperda (Sf9) cells is distinctive from that produced in Escherichia coli

Kueh CL, Yong CY, Masoomi Dezfooli S, Bhassu S, Tan SG, Tan WS

Biotechnol Prog, 2017 Mar;33(2):549-557.
PMID: 27860432 DOI: 10.1002/btpr.2409

Macrobrachium rosenbergii nodavirus (MrNV) is a virus native to giant freshwater prawn. Recombinant MrNV capsid protein has been produced in Escherichia coli, which self-assembled into virus-like particles (VLPs). However, this recombinant protein is unstable, degrading and forming heterogenous VLPs. In this study, MrNV capsid protein was produced in insect Spodoptera frugiperda (Sf9) cells through a baculovirus system. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) revealed that the recombinant protein produced by the insect cells self-assembled into highly stable, homogenous VLPs each of approximately 40 nm in diameter. Enzyme-linked immunosorbent assay (ELISA) showed that the VLPs produced in Sf9 cells were highly antigenic and comparable to those produced in E. coli. In addition, the Sf9 produced VLPs were highly stable across a wide pH range (2-12). Interestingly, the Sf9 produced VLPs contained DNA of approximately 48 kilo base pairs and RNA molecules. This study is the first report on the production and characterization of MrNV VLPs produced in a eukaryotic system. The MrNV VLPs produced in Sf9 cells were about 10 nm bigger and had a uniform morphology compared with the VLPs produced in E. coli. The insect cell production system provides a good source of MrNV VLPs for structural and immunological studies as well as for host-pathogen interaction studies. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:549-557, 2017.

Matched MeSH terms: Cloning, Molecular/methods
Molecular cloning and characterization of an atypical butyrylcholinesterase-like protein in zebrafish

Tan KS, Zhang Y, Liu L, Li S, Zou X, Zeng W, et al.

Comp Biochem Physiol B Biochem Mol Biol, 2021 03 02;255:110590.
PMID: 33662568 DOI: 10.1016/j.cbpb.2021.110590

Cholinesterases act as bio scavengers to clear organophosphorus (OP) compounds and prodrugs. The butyrylcholinesterase (BChE) gene has been found in several types of teleost fish but this gene has yet to be identified in cyprinid fish. Indeed, BChE homologs have not been found in the zebrafish (Danio rerio) genomic database. Here, we demonstrate that BChE activity is present in zebrafish, in line with other groups' findings. Using in-gel native-PAGE enzymatic activity staining and LC-MS/MS technique, an atypical BChE-like protein was identified in zebrafish. The si:ch211-93f2.1 gene was cloned, and His-tagged recombinant protein was expressed using the Pichia yeast system. The purified protein (molecular weight ~ 180 kDa) showed BChE activity, and degraded acetylcholinesterase (ACh) at a higher rate than BCh. However, phylogram analysis shows that this novel cholinesterase shared an evolutionary origin with carboxylic esterase rather than BChE. The zebrafish BChE-like protein shares structural characteristics with cholinesterase and carboxylesterase. The 2-arachidonoylglycerol (2-AG), nicosulfuron, and triacetin exhibited a higher binding affinity to the zebrafish BChE-like protein than BCh and ACh. With the identification of BChE-like protein in zebrafish, this study could shed light on the origin of BChE and may contribute towards the development of a BChE knockout zebrafish model for sensitive drug or toxin screening.

Matched MeSH terms: Cloning, Molecular*
Six isoforms of cardiotoxin in malayan spitting cobra (Naja naja sputatrix) venom: cloning and characterization of cDNAs

Jeyaseelan K, Armugam A, Lachumanan R, Tan CH, Tan NH

Biochim. Biophys. Acta, 1998 Apr 10;1380(2):209-22.
PMID: 9565688

Cardiotoxins are the most abundant toxin components of cobra venom. Although many cardiotoxins have been purified and characterized by amino acid sequencing and other pharmacological and biochemical studies, to date only five cardiotoxin cDNAs from Taiwan cobra (Naja naja atra), three cDNAs from Chinese cobra (Naja atra) and two more of uncertain origin (either Chinese or Taiwan cobra) have been reported. In this paper we show the existence of four isoforms of cardiotoxin by protein analysis and nine cDNA sequences encoding six isoforms of cardiotoxins (CTX 1-3, 4a, 4b and 5) from N. n. sputatrix by cDNA cloning. This forms the first report on the cloning and characterization of several cardiotoxin genes from a single species of a spitting cobra. The cDNAs encoding these isoforms, obtained by reverse transcription-polymerase chain reaction (RT-PCR), were subsequently expressed in Escherichia coli. The native and recombinant cardiotoxins were first characterized by Western blotting and N-terminal protein sequencing. These proteins were also found to have different levels of cytolytic activity on cultured baby hamster kidney cells. Four of the isoforms (CTX 1, 2, 4 and 5) are unique to N. n. sputatrix, with CTX 2 being the most abundant species constituting about 50% of the total cardiotoxins. The isoform CTX 3 (20% constitution) is highly homologous to the cardiotoxins of N. n. atra and N. n. naja, indicating that it may be universally present in all Naja naja subspecies. Our studies suggest that the most hydrophilic isoform (CTX 5) could have evolved first followed by the hydrophobic isoforms (CTX 1, 2, 3 and 4). We also speculate that Asiatic cobras could be the modern descendants of the African and Egyptian counterparts.

Matched MeSH terms: Cloning, Molecular
Fulltext Subtype distribution of Blastocystis isolates in Sebha, Libya

Abdulsalam AM, Ithoi I, Al-Mekhlafi HM, Al-Mekhlafi AM, Ahmed A, Surin J

PLoS One, 2013;8(12):e84372.
PMID: 24376805 DOI: 10.1371/journal.pone.0084372

BACKGROUND: Blastocystis is a genetically diverse and a common intestinal parasite of humans with a controversial pathogenic potential. This study was carried out to identify the Blastocystis subtypes and their association with demographic and socioeconomic factors among outpatients living in Sebha city, Libya.
METHODS/FINDINGS: Blastocystis in stool samples were cultured followed by isolation, PCR amplification of a partial SSU rDNA gene, cloning, and sequencing. The DNA sequences of isolated clones showed 98.3% to 100% identity with the reference Blastocystis isolates from the Genbank. Multiple sequence alignment showed polymorphism from one to seven base substitution and/or insertion/deletion in several groups of non-identical nucleotides clones. Phylogenetic analysis revealed three assemblage subtypes (ST) with ST1 as the most prevalent (51.1%) followed by ST2 (24.4%), ST3 (17.8%) and mixed infections of two concurrent subtypes (6.7%).
BLASTOCYSTIS: ST1 infection was significantly associated with female (P = 0.009) and low educational level (P = 0.034). ST2 was also significantly associated with low educational level (P= 0.008) and ST3 with diarrhoea (P = 0.008).
CONCLUSION: Phylogenetic analysis of Libyan Blastocystis isolates identified three different subtypes; with ST1 being the predominant subtype and its infection was significantly associated with female gender and low educational level. More extensive studies are needed in order to relate each Blastocystis subtype with clinical symptoms and potential transmission sources in this community.

Matched MeSH terms: Cloning, Molecular
Expression and the antigenicity of recombinant coat proteins of tungro viruses expressed in Escherichia coli

Yee SF, Chu CH, Poili E, Sum MSH

J Virol Methods, 2017 02;240:69-72.
PMID: 27923590 DOI: 10.1016/j.jviromet.2016.12.001

Rice tungro disease (RTD) is a recurring disease affecting rice farming especially in the South and Southeast Asia. The disease is commonly diagnosed by visual observation of the symptoms on diseased plants in paddy fields and by polymerase chain reaction (PCR). However, visual observation is unreliable and PCR can be costly. High-throughput as well as relatively cheap detection methods are important for RTD management for screening large number of samples. Due to this, detection by serological assays such as immunoblotting assays and enzyme-linked immunosorbent assay are preferred. However, these serological assays are limited by lack of continuous supply of antibodies as reagents due to the difficulty in preparing sufficient purified virions as antigens. This study aimed to generate and evaluate the reactivity of the recombinant coat proteins of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV) as alternative antigens to generate antibodies. The genes encoding the coat proteins of both viruses, RTBV (CP), and RTSV (CP1, CP2 and CP3) were cloned and expressed as recombinant fusion proteins in Escherichia coli. All of the recombinant fusion proteins, with the exception of the recombinant fusion protein of the CP2 of RTSV, were reactive against our in-house anti-tungro rabbit serum. In conclusion, our study showed the potential use of the recombinant fusion coat proteins of the tungro viruses as alternative antigens for production of antibodies for diagnostic purposes.

Matched MeSH terms: Cloning, Molecular
Identification of a new polyhydroxyalkanoate (PHA) producer Aquitalea sp. USM4 (JCM 19919) and characterization of its PHA synthase

Ng LM, Sudesh K

J Biosci Bioeng, 2016 Nov;122(5):550-557.
PMID: 27132174 DOI: 10.1016/j.jbiosc.2016.03.024

Aquitalea sp. USM4 (JCM 19919) was isolated from a freshwater sample at Lata Iskandar Waterfall in Perak, Malaysia. It is a rod-shaped, gram-negative bacterium with high sequence identity (99%) to Aquitalea magnusonii based on 16S rRNA gene analysis. Aquitalea sp. USM4 also possessed a PHA synthase gene (phaC), which had amino acid sequence identity of 77-78% to the PHA synthase of Chromobacterium violaceum ATCC12472 and Pseudogulbenkiania sp. NH8B. PHA biosynthesis results showed that wild-type Aquitalea sp. USM4 was able to accumulate up to 1.5 g/L of poly(3-hydroxybutyrate), [P(3HB)]. The heterologous expression of the PHA synthase gene of Aquitalea sp. USM4 (phaCAq) in Cupriavidus necator PHB(-)4 had resulted in PHA accumulation up to 3.2 g/L of P(3HB). It was further confirmed by (1)H nuclear magnetic resonance (NMR) analysis that Aquitalea sp. USM4 and C. necator PHB(-)4 transformant were able to produce PHA containing 3-hydroxyvalerate (3HV), 4-hydroxybutyrate (4HB) and 3-hydroxy-4-methylvalerate (3H4MV) monomers from suitable precursor substrates. Interestingly, relatively high PHA synthase activity of 863 U/g and 1402 U/g were determined in wild-type Aquitalea sp. USM4 and C. necator PHB(-)4 transformant respectively. This is the first report on the member of genus Aquitalea as a new PHA producer as well as in vitro and in vivo characterization of a novel PHA synthase from Aquitalea sp. USM4.

Matched MeSH terms: Cloning, Molecular
Fulltext Characterization of the highly active polyhydroxyalkanoate synthase of Chromobacterium sp. strain USM2

Bhubalan K, Chuah JA, Shozui F, Brigham CJ, Taguchi S, Sinskey AJ, et al.

Appl Environ Microbiol, 2011 May;77(9):2926-33.
PMID: 21398494 DOI: 10.1128/AEM.01997-10

The synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolated Chromobacterium sp. USM2 (PhaC(Cs)). PhaC(Cs) showed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. An in vitro assay of recombinant PhaC(Cs) expressed in Escherichia coli showed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 ± 80 U/g) than that of the synthase from the model strain C. necator (307 ± 24 U/g). Specific activity using a Strep2-tagged, purified PhaC(Cs) was 238 ± 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC from C. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation in Escherichia coli expressing PhaC(Cs) of up to 76 ± 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaC(Cs) is a naturally occurring, highly active PHA synthase with superior polymerizing ability.

Matched MeSH terms: Cloning, Molecular
An Application of pET SUMO Protein Expression System in Escherichia coli: Cloning, Expression, Purification, and Characterisation of Native Kras4BG12V Oncoprotein

Tan MS, Teh YH, Ho KL, Stanslas J

Protein J, 2020 02;39(1):54-61.
PMID: 31620959 DOI: 10.1007/s10930-019-09872-1

Being an important regulator of cell growth and survival, a point mutation at glycine-12 residue of Kras4B to valine (V), renders Kras4BG12V oncogenic. Kras4B recombinant protein is used as a bait to fish its potential ligands in the attempt of drugging this oncoprotein and to validate its pharmacologically relevant ligand in protein-ligand interaction studies. Nevertheless, synthesis of Kras4B recombinant protein is challenging as it was reported being susceptible to aggregation into inclusion bodies in the bacterial host, resulting in a poor yield of recombinant protein. Here, we describe a novel method to produce native Kras4BG12V protein by using pET SUMO protein expression system as a solution to the formation of inclusion bodies. Kras4BG12V oncogene was cloned into pET SUMO vector, followed by a 12 h chemically induced protein expression in Escherichia coli at 20 °C. Native Kras4BG12V protein was produced in a series of protein purification steps involving immobilised nickel ion-affinity column chromatography, SUMO fusion protein and polyhistidine tag removal, and size exclusion column chromatography. The identity of the purified Kras4BG12V protein was validated by immunoblot analysis. The purified protein exhibited self-dimerising, indicating that the purified protein structurally resembles Kras4B. Its physical interaction with 4,6-dichloro-2-methyl-3-aminoethyl-indole (DCAI), a known binder of Kras4B, confirms the identity of the purified protein as Kras4BG12V. The native Kras4BG12V protein was successfully purified in a substantial amount by using the pET SUMO protein expression system.

Matched MeSH terms: Cloning, Molecular
Comparison of myeloperoxidase detection by flow cytometry using two different clones of monoclonal antibodies

Leong CF, Kalaichelvi AV, Cheong SK, Hamidah NH, Rahman J, Sivagengei K

Malays J Pathol, 2004 Dec;26(2):111-6.
PMID: 16329563

Myeloperoxidase (MPO) is present in azurophilic granules which appear in the promyelocyte stage of differentiation and is expressed in granulomonocytic cells. MPO is usually detected by cytochemistry. The demonstration of peroxidase in at least 3% of bone marrow blasts defines an acute leukaemia as acute myeloblastic leukaemia (AML). MPO is important in distinguishing acute myeloblastic leukaemia (AML) from acute lymphoblastic leukaemia (ALL). It is difficult to diagnose AML with minimal evidence of myeloid differentiation (AML- M0) by conventional light microscopy. However, these AML-M0 blasts can be detected by monoclonal antibodies. Anti-MPO recognizes the enzymatically inactive precursor forms of MPO. There are a few commercially available monoclonal antibodies against MPO. In this study, we evaluated two monoclonal antibodies against MPO from different commercial sources.

Matched MeSH terms: Cloning, Molecular

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