Papers by Montarop Yamabhai
PLOS Pathogens, May 28, 2019
Indonesian Journal of Biotechnology, Feb 13, 2018
Ochratoxin A (OTA) is a mycotoxin commonly found in agricultural products and can accumulate in t... more Ochratoxin A (OTA) is a mycotoxin commonly found in agricultural products and can accumulate in the blood and ssues a er contaminated food is consumed. Recombinant single-chain an body fragments (scFv) against OTA were selected from phage display libraries. A er one round of biopanning against BSA-conjugated OTA (OTA-BSA), 52 and 6 phage clones displaying scFv an bodies were isolated from human (Yamo I.3) and rabbit (Bozmix I.2) libraries. Two phage clones (one from each library, i.e. yOTA1e3 and bOTA2a9) showed binding to free toxin by compe ve ELISA. The soluble scFv an bodies were produced by superinfec ng phage clones into Escherichia coli suppressor strain HB2151. The scFv genes from these two phage clones were sub-cloned into pKP300∆III vectors to generate scFv-AP fusions. The binding affinity (IC 50) of an bodies derived from the human library were higher than those from the rabbit library. The binding property of recombinant an bodies in the form of scFv-AP was be er than those in soluble scFv form. Cross-reac vity analysis indicated that the two recombinant an bodies did not cross-react with other soluble toxins, namely AFB1, DON, ZEN, and FB. The ability to use the recombinant scFv-AP to detect contaminated toxins in an agricultural product (corn) was demonstrated. These an bodies can be used as a template for further engineering and op miza on for the detec on of contaminated OTAs in the future.
Journal of Biotechnology, 2008
Bacillus spp. are Gram-positive bacteria that secrete a large number of extracellular proteins of... more Bacillus spp. are Gram-positive bacteria that secrete a large number of extracellular proteins of industrial relevance. In this report, three Bacillus extracellular hydrolytic enzymes, i.e., alpha-amylase, mannanase and chitinase, were cloned and over-expressed in Gram-negative Escherichia coli. We found that both the native signal peptides and that of E. coli outer membrane protein, OmpA, could be used to direct the secretion of the recombinant enzymes. The expressed enzymes were observed as clearing zones on agar plates or in zymograms. Determination of enzyme activities in different cell compartments suggested that the ability of the enzymes to be secreted out into the culture medium depends on the time of induction, the type of the signal peptides and the molecular mass of the enzymes. After overnight induction, most of the enzyme activities (85-96%) could be harvested from the culture supernatant. Our results suggest that various signal peptides of Bacillus spp. can be recognized by the E. coli secretion machinery. It seems possible that other enzymes with similar signal peptide could be secreted equally well in E. coli expression systems. Thus, our finding should be able to apply for cloning and extracellular production of other Bacillus hydrolytic enzymes as well as other proteins.
Bioresource Technology, 2010
Chitinase (EC 3.2.1.14) is an enzyme with multiple industrial applications. These include bioconv... more Chitinase (EC 3.2.1.14) is an enzyme with multiple industrial applications. These include bioconversion of chitin waste, a highly resistant and abundant biopolymer from crustacean food industry, into glucosamine and chito-oligosaccharide value-added products. This paper reports on the expression of endochitinase (ChiA) from Bacillus licheniformis strain DSM8785 in E. coli and characterization of the recombinant enzyme. Recombinant ChiA could efficiently convert colloidal chitin to N-acetyl glucosamine and chitobiose at pH 4.0, 6.0 and 9.0 at 50 degrees C and retained its activity up to 3days under these conditions, suggesting that this enzyme is suitable for bioconversion of chitin waste.
Scientific Reports, Jun 28, 2023
The glutamine synthetase (GS)-based Chinese hamster ovary (CHO) selection system is an attractive... more The glutamine synthetase (GS)-based Chinese hamster ovary (CHO) selection system is an attractive approach to efficiently identify suitable clones in the cell line generation process for biologics manufacture, for which GS-knockout (GS-KO) CHO cell lines are commonly used. Since genome analysis indicated that there are two GS genes in CHO cells, deleting only 1 GS gene could potentially result in the activation of other GS genes, consequently reducing the selection efficiency. Therefore, in this study, both GS genes identified on chromosome 5 (GS5) and 1 (GS1) of CHO-S and CHO-K1, were deleted using CRISPR/Cpf1. Both single and double GS-KO CHO-S and K1 showed robust glutaminedependent growth. Next, the engineered CHO cells were tested for their efficiency of selection of stable producers of two therapeutic antibodies. Analysis of pool cultures and subclones after a single round of 25 µM methionine sulfoxinime (MSX) selection indicated that for CHO-K1 the double GS5,1-KO was more efficient as in the case of a single GS5-KO the GS1 gene was upregulated. In CHO-S, on the other hand, with an autologously lower level of expression of both variants of GS, a single GS5-KO was more robust and already enabled selection of high producers. In conclusion, CRISPR/ Cpf1 can be efficiently used to knock out GS genes from CHO cells. The study also indicates that for the generation of host cell lines for efficient selection, the initial characterisation of expression levels of the target gene as well as the identification of potential escape mechanisms is important.
Journal of Biotechnology, May 1, 2007
A gene of-galactosidase from Bacillus circulans ATCC 31382 was cloned and sequenced on the basis ... more A gene of-galactosidase from Bacillus circulans ATCC 31382 was cloned and sequenced on the basis of N-terminal and internal peptide sequences isolated from a commercial enzyme preparation, Biolacta Ò. Using the cloned gene, recombinant-galactosidase and its deletion mutants were overexpressed as His-tagged proteins in Escherichia coli cells and the enzymes expressed were characterized.
Human monoclonal antibodies against Rabies virus were selected from non-immunized human scFv libr... more Human monoclonal antibodies against Rabies virus were selected from non-immunized human scFv library (YAMO-I library) and immunized library (Yamo-Rb library) by using phage display technology. The biopanning was performed for 2-5 rounds by using two types of inactivated rabies vaccines as targets. These are purified vero cell rabies vaccine (PVRV) and purified chick embryo cell vaccine (PCEC). A total of 14 positive clones from various method of biopanning that can bind to rabies, i.e.; IRA7c,
Applied Biochemistry and Biotechnology, Jun 27, 2013
hCNT1 and hCNT2 mediate concentrative (Na ؉linked) cellular uptake of nucleosides and nucleoside ... more hCNT1 and hCNT2 mediate concentrative (Na ؉linked) cellular uptake of nucleosides and nucleoside drugs by human cells and tissues. The two proteins (650 and 658 residues, 71 kDa) are 72% identical in sequence and contain 13 putative transmembrane helices (TMs). When produced in Xenopus oocytes, recombinant hCNT1 is selective for pyrimidine nucleosides (system cit), whereas hCNT2 is selective for purine nucleosides (system cif). Both transport uridine. We have used (i) chimeric constructs between hCNT1 and hCNT2, (ii) sequence comparisons with a newly identified broad specificity concentrative nucleoside transporter (system cib) from Eptatretus stouti, the Pacific hagfish (hfCNT), and (iii) site-directed mutagenesis of hCNT1 to identify two sets of adjacent residues in TMs 7 and 8 of hCNT1 (Ser 319 /Gln 320 and Ser 353 /Leu 354) that, when converted to the corresponding residues in hCNT2 (Gly 313 /Met 314 and Thr 347 /Val 348), changed the specificity of the transporter from cit to cif. Mutation of Ser 319 in TM 7 of hCNT1 to Gly enabled transport of purine nucleosides, whereas concurrent mutation of Gln 320 to Met (which had no effect on its own) augmented this transport. The additional mutation of Ser 353 to Thr in TM 8 converted hCNT1/ S319G/Q320M, from cib to cif, but with relatively low adenosine transport activity. Additional mutation of Leu 354 to Val (which had no effect on its own) increased the adenosine transport capability of hCNT1/S319G/ Q320M/S353T, producing a full cif-type transporter phenotype. On its own, the S353T mutation converted hCNT1 into a transporter with novel uridine-selective transport properties. Helix modeling of hCNT1 placed Ser 319 (TM 7) and Ser 353 (TM 8) within the putative substrate translocation channel, whereas Gln 320 (TM 7) and Leu 354 (TM 8) may exert their effects through altered helix packing.
Medical Oncology, Sep 29, 2022
Protein Expression and Purification, May 1, 2012
Glutaminase or L-glutamine aminohydrolase (EC 3.5.1.2) is an enzyme that catalyzes the formation ... more Glutaminase or L-glutamine aminohydrolase (EC 3.5.1.2) is an enzyme that catalyzes the formation of glutamic acid and ammonium ion from glutamine. This enzyme functions in cellular metabolism of every organism by supplying nitrogen required for the biosynthesis of a variety of metabolic intermediates, while glutamic acid plays a role in both sensory and nutritional properties of food. So far there have been only a few reports on cloning, expression and characterization of purified glutaminases. Microbial glutaminases are enzymes with emerging potential in both the food and the pharmaceutical industries. In this research a recombinant glutaminase from Bacillus licheniformis (GlsA) was expressed in Escherichia coli, under the control of a ptac promoter. The recombinant enzyme was tagged with decahistidine tag at its C-terminus and could be conveniently purified by one-step immobilized metal affinity chromatography (IMAC) to apparent homogeneity. The enzyme could be induced for efficient expression with IPTG, yielding approximately 26,000 units from 1-l shake flask cultures. The enzyme was stable at 30°C and pH 7.5 for up to 6 h, and could be used efficiently to increase glutamic acid content when protein hydrolysates from soy and anchovy were used as substrates. The study demonstrates an efficient expression system for the production and purification of bacterial glutaminase. In addition, its potential application for bioconversion of glutamine to flavor-enhancing glutamic acid has been demonstrated.
Journal of Biomedical Materials Research Part A, May 24, 2023
Despite accumulating evidences have demonstrated the potential of collagen and chitosan on tissue... more Despite accumulating evidences have demonstrated the potential of collagen and chitosan on tissue repair, it remains unclear on their combination effects. Here, we examined the regenerative effects of single collagen, chitosan and their mixture on fibroblasts and endothelial cells at cellular levels. The results showed that fibroblast responses, as indicated by high proliferative rate, increased spheroid diameter and migrated area existing from spheroid edge, and decreased wound area, were significantly promoted by either collagen or chitosan stimulation. Similarly, both collagen and chitosan resulted in increased endothelial cell proliferation and migration with accelerated tube‐like network formation and upregulated VE‐cadherin expression, although collagen strongly provided this effect. While the 1:1 mixture (100:100 μg/mL of chitosan to collagen) treatment caused a reduction in fibroblast viability, the lower ratio of chitosan (1:10 mixture; 10:100 μg/mL) did not produce any impact on both fibroblast and endothelial cell viabilities. The 1:10 mixture also significantly enhanced the additional effects on fibroblast responses and angiogenic activities as shown by higher endothelial growth, proliferation and migration with accelerated capillary‐like network formation than those treated with the single substance. Further investigation of signaling proteins found that collagen significantly increased expressions of p‐Fak, p‐Akt and Cdk5 whereas chitosan upregulated p‐Fak and Cdk5 expressions. Comparing to the single treatments, p‐Fak, p‐Akt and Cdk5 were higher expressed in the 1:10 mixture. These observations indicate that proper collagen‐chitosan mixture provides the combination effects on fibroblast responses and angiogenic activities when a high concentration of collagen is used, possibly through Fak/Akt and Cdk5 signaling pathways. Therefore, this study helps to define the clinical use of collagen and chitosan as promising biomaterials for tissue repair.
Biochemical and Biophysical Research Communications, Sep 1, 2022
Toxicon, Jul 1, 2021
Snakebite is an important public health problem in tropical and subtropical regions. Macrovipera ... more Snakebite is an important public health problem in tropical and subtropical regions. Macrovipera lebetina is one of the most dangerous snakes in Iran. Envenoming by this snake can lead to respiratory distress, heart attack, bleeding, and death. The specific treatment available is immunized equine serum, which has several side effects like serum sickness. Nowadays, single-chain fragment variable antibodies (scFvs) are one of the fast growing classes of monoclonal antibodies, which are suggested for treatment of envenoming. This study aimed to achieve a fully human scFv antibody against M. lebetina venom from human non-immune library. In this study, scFvs against M. lebetina venom were isolated by phage display technique. Using three rounds of biopanning, two specific scFvs (C37 and C69) with the highest affinity were selected. The selected scFvs purified by nickel affinity chromatography. The specific binding of purified antibodies were confirmed by enzyme-linked immunosorbent assay. The LD50 as well as HD50 concentration of the crude venom were obtained to be 45 μg and 120 μg/ml, respectively. C69 neutralized 48% of the hemolysis activity of M. lebetina venom and C37 survived 66% of mice after 115 min of envenoming. Taken together, the results indicate the potential of human non-immune libraries for selection of functional antibodies against M. lebetina venom.
Carbohydrate Polymers, Apr 1, 2018
Bioconversion of chitosan into chito-oligosaccharides (CHOS) using family 46 chitosanase from Bac... more Bioconversion of chitosan into chito-oligosaccharides (CHOS) using family 46 chitosanase from Bacillus subtilis (BsCsn46A)
Applied Microbiology and Biotechnology, Mar 28, 2023
Applied Microbiology and Biotechnology
Efficient selection and production of antibody fragments in microbial systems remain to be a chal... more Efficient selection and production of antibody fragments in microbial systems remain to be a challenging process. To optimize microbial production of single-chain variable fragments (scFvs), we have chosen five model targets, 1) a hapten, Zearalenone (ZEN) mycotoxin, along with infectious agents 2) rabies virus, 3) Propionibacterium acnes, 4) Pseudomonas aeruginosa, and a cancer cell 5) acute myeloid leukemia cell line (HL-60). The scFv binders were affinity selected from a non-immunized human phage display scFv antibody library and genetically fused to the N-terminus of emerald green fluorescent protein (EmGFP). The scFv-EmGFP fusion constructs were subcloned into an expression vector, under the control of T7 promoter, C-terminally tagged with hexa-histidine and expressed in different Escherichia coli (E. coli) hosts. This enabled the detection of cells that expressed the correct scFv-EmGFP fusion, termed fluorobody, via bright fluorescent signal in the cytoplasm. Among the three E...
Micromachines
Genetically-modified monoclonal cell lines are currently used for monoclonal antibody (mAbs) prod... more Genetically-modified monoclonal cell lines are currently used for monoclonal antibody (mAbs) production and drug development. The isolation of single transformed cells is the main hindrance in the generation of monoclonal lines. Although the conventional limiting dilution method is time-consuming, laborious, and skill-intensive, high-end approaches such as fluorescence-activated cell sorting (FACS) are less accessible to general laboratories. Here, we report a bench-top approach for isolating single Chinese hamster ovary (CHO) cells using an adapted version of a simple microwell-based microfluidic (MBM) device previously reported by our group. After loading the cell suspension to the device, the electrostatically trapped cells can be viewed under a microscope and transferred using a micropipette for further clone establishment. Compared to the conventional method, the invented approach provided a 4.7-fold increase in the number of single cells isolated per round of cell loading and ...
Biochemical and Biophysical Research Communications
Scientific Reports, 2022
To improve the potency of Heptamethine cyanines (Hcyanines) in cancer research, we designed and s... more To improve the potency of Heptamethine cyanines (Hcyanines) in cancer research, we designed and synthesized two novel Hcyanines based theranostic probes, IR794-Morph and IR794-Morph-Mpip, to enhance cancer cell internalization and targeting. In acidic conditions that resemble to tumour environment, both IR794 derivatives exhibited broad NIR absorption band (704‒794 nm) and fluorescence emission (798‒828 nm) that is suitable for deep seated tumour imaging. Moreover, in vitro study revealed that IR794-Morph-Mpip exhibited better cancer targetability towards various cancer cell lines under physiological and slightly acidic conditions compared to normal cells. IR794-Morph-Mpip was fast internalized into the cancer cells within the first 5 min and mostly localized in lysosomes and mitochondria. In addition, the internalized signal was brighter when the cells were in the hypoxic environment. Furthermore, cellular uptake mechanism of both IR794 dyes, investigated via flow cytometry, reveal...
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Papers by Montarop Yamabhai