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A mouse IgM monoclonal antibody recognizes breast and colon cancer

Bakar AF, Alitheen NB, Keong YS, Hamid M, Ali SA, Ali AM

Hybridoma (Larchmt), 2009 Jun;28(3):199-203.
PMID: 19519247 DOI: 10.1089/hyb.2007.0531

Hybridoma clone C3A8, which is a fusion product between splenic lymphocytes of Balb/c mice immunized with MCF7 breast carcinoma cells and SP2/0 myelomas, was produced and characterized. A stable clone that secreted IgM monoclonal antibody (MAb) with kappa light chain was obtained through limiting dilutions. Cell-ELISA screening, flow cytometry analysis, and immunofluorescence staining revealed that the MAb C3A8 had bound specifically and strongly to MCF7 and HT29 but cross reacted weakly or not on HeLa cell line. The MAb C3A8 reacted positively with paraffin-embedded tissues of human breast and colon cancers but there were no positive reactions on normal tissues. Western blot analysis showed the MAb recognized a 55 kDa protein, which was present in the extract of MCF7 and HT29 cell lines. Our results demonstrated that MAb C3A8 could be used for basic and clinical research of breast and colon cancers.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Fulltext Enhanced production, purification, characterization and mechanism of action of salivaricin 9 lantibiotic produced by Streptococcus salivarius NU10

Barbour A, Philip K, Muniandy S

PLoS One, 2013;8(10):e77751.
PMID: 24147072 DOI: 10.1371/journal.pone.0077751

BACKGROUND: Lantibiotics are small lanthionine-containing bacteriocins produced by lactic acid bacteria. Salivaricin 9 is a newly discovered lantibiotic produced by Streptococcus salivarius. In this study we present the mechanism of action of salivaricin 9 and some of its properties. Also we developed new methods to produce and purify the lantibiotic from strain NU10.
METHODOLOGY/PRINCIPAL FINDINGS: Salivaricin 9 was found to be auto-regulated when an induction assay was applied and this finding was used to develop a successful salivaricin 9 production system in liquid medium. A combination of XAD-16 and cation exchange chromatography was used to purify the secondary metabolite which was shown to have a molecular weight of approximately 3000 Da by SDS-PAGE. MALDI-TOF MS analysis indicated the presence of salivaricin 9, a 2560 Da lantibiotic. Salivaricin 9 is a bactericidal molecule targeting the cytoplasmic membrane of sensitive cells. The membrane permeabilization assay showed that salivaricin 9 penetrated the cytoplasmic membrane and induced pore formation which resulted in cell death. The morphological changes of test bacterial strains incubated with salivaricin 9 were visualized using Scanning Electron Microscopy which confirmed a pore forming mechanism of inhibition. Salivaricin 9 retained biological stability when exposed to high temperature (90-100°C) and stayed bioactive at pH ranging 2 to 10. When treated with proteinase K or peptidase, salivaricin 9 lost all antimicrobial activity, while it remained active when treated with lyticase, catalase and certain detergents.
CONCLUSION: The mechanism of antimicrobial action of a newly discovered lantibiotic salivaricin 9 was elucidated in this study. Salivaricin 9 penetrated the cytoplasmic membrane of its targeted cells and induced pore formation. This project has given new insights on lantibiotic peptides produced by S. salivarius isolated from the oral cavities of Malaysian subjects.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Paediatric gastroenteritis in the eastern Malaysian state of Sarawak: an epidemiological and clinical study

Barker RA, Maxwell PH, Hong CP, Cordery MC, Chrystie IL

Trans R Soc Trop Med Hyg, 1988;82(6):898-901.
PMID: 2855768

Over a period of 2 months, 35 of 69 (51%) cases of juvenile diarrhoea studied in eastern Malaysia were associated with rotavirus excretion; rotavirus associated diarrhoea occurred most commonly in the 6-24 month age group. Polyacrylamide gel electrophoresis (PAGE) of genome ribonucleic acid showed that only 4 rotavirus electropherotypes could be detected. Of those, 2 predominated and 2 were detected only once each; one of these may have been a reassortment of the two predominant electropherotypes. Analysis of the clinical features of patients excreting rotavirus subgroup 1 or 2, determined by PAGE, demonstrated that rotavirus subgroup 1 was associated with more hypotonic dehydration and need for intravenous therapy: lethargy was significantly more common among those excreting rotavirus subgroup 2.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Fulltext Cross-reactivity of Malaysian rat cytomegalovirus strains with its human counterpart

Camalxaman SN, Zeenathul NA, Quah YW, Loh HS, Zuridah H, Sheikh-Omar AR, et al.

Trop Biomed, 2011 Dec;28(3):661-7.
PMID: 22433897 MyJurnal

This study probes into the prospect of cross-reactivity of HCMV with RCMV which has not been acknowledged to date. We describe the uncovering of a protein with an estimated size of between 61-68 kDa from local RCMV strains which reacted with HCMV positive sera. Our findings are a first disclosure of a plausible immunological cross-reactivity between RCMV with its human counterpart which grounds substantial interest implying existence of conserved determinants between rat and human CMV polypeptides. The cross-reactive protein most likely represents an enveloped glycoprotein, though the precise identification and its degree of similarity needs to be evidently defined and further elucidated in forthcoming experiments.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Detection of IgM and IgG antibodies to Cryptococcus neoformans proteins in blood donors and HIV patients with active cryptococcosis

Chai HC, Tay ST

Mycoses, 2009 Mar;52(2):166-70.
PMID: 18643920 DOI: 10.1111/j.1439-0507.2008.01549.x

The serological responses to Cryptococcus neoformans proteins of blood donors and HIV patients with active cryptococcosis from a tropical region were investigated in this study. Exposure to C. neoformans, an organism ubiquitous in the environment, contributes to the antibody responses observed in the blood donors. IgG responses to cryptococcal proteins were stronger than IgM responses in most sera tested in this study. A 53-kDa cryptococcal protein fragment was identified as the most immunoreactive protein on the IgM immunoblots of both blood donors and patients. Overall, there was no obvious difference in IgG responses of patients when compared with blood donors. Some immunogenic protein fragments (27.5, 76, 78 and 91.5 kDa) were detected at least two times more frequently on IgM immunoblots of patients compared with those of blood donors. It is yet to be investigated whether the proteins identified in this study may have any potential to be used as biomarker for cryptococcosis.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Myosin heavy chain isoforms expression, calpain system and quality characteristics of different muscles in goats

Chaosap C, Sitthigripong R, Sivapirunthep P, Pungsuk A, Adeyemi KD, Sazili AQ

Food Chem, 2020 Aug 15;321:126677.
PMID: 32247180 DOI: 10.1016/j.foodchem.2020.126677

Myosin heavy chain (MHC) isoforms in goat muscles and their possible relationships with meat quality have not been fully elucidated. This study characterized the MHC isoforms in different caprine muscles using sodium dodecyl sulphate glycerol gel electrophoresis (SDS-GGE). The relationships between MHC isoforms, calpain systems and meat quality characteristics of different muscles in goats were examined. Four muscles, namely infraspinatus (IF), longissimus dorsi (LD), psoas major (PM) and supraspinatus (SS) were obtained from ten Boer crossbred bucks (7-10 months old; 26.5 ± 3.5 kg, BW). The percentages of MHC I, MHC IIa and MHC IIx in SS, IF, PM and LD were 47.2, 38.3, 32.1, 11.9; 28.0, 42.1, 33.0, 36.4; and 24.8, 19.6, 34.9 and 51.7, respectively. IF and SS had higher levels of calpastatin, total collagen and insoluble collagen contents than did PM and LD. PM had longer sarcomere length than did other muscles. LD had higher collagen solubility, troponin-T degradation products and glycogen content than did other muscles. These results infer that variable fiber-type composition could account partially for the differences in the physicochemical properties of goat muscles.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Fulltext Construction and heterologous expression of a truncated Haemagglutinin (HA) protein from the avian influenza virus H5N1 in Escherichia coli

Chee Wei T, Nurul Wahida AG, Shaharum S

Trop Biomed, 2014 Dec;31(4):792-801.
PMID: 25776606 MyJurnal

Malaysia first reported H5N1 poultry case in 2004 and subsequently outbreak in poultry population in 2007. Here, a recombinant gene encoding of peptide epitopes, consisting fragments of HA1, HA2 and a polybasic cleavage site of H5N1 strain Malaysia, was amplified and cloned into pET-47b(+) bacterial expression vector. DNA sequencing and alignment analysis confirmed that the gene had no alteration and in-frame to the vector. Then, His-tagged truncated HA protein was expressed in Escherichia coli BL21 (DE3) under 1 mM IPTG induction. The protein expression was optimized under a time-course induction study and further purified using Ni-NTA agarose under reducing condition. Migration size of protein was detected at 15 kDa by Western blot using anti-His tag monoclonal antibody and demonstrated no discrepancy compared to its calculated molecular weight.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Fulltext Fabrication and Evaluation of Transdermal Microneedles for a Recombinant Human Keratinocyte Growth Factor

Chellathurai MS, Ling VWT, Palanirajan VK

Turk J Pharm Sci, 2021 Feb 25;18(1):96-103.
PMID: 33634684 DOI: 10.4274/tjps.galenos.2020.21033

Objectives: Microneedle transdermal patches are a combination of hypodermic needles and transdermal patches used to overcome the individual limitations of both injections and patches. The objective of this study was to design a minimally invasive, biodegradable polymeric recombinant human keratinocyte growth factor (rHuKGF) microneedle array and evaluate the prepared biodegradable microneedles using in vitro techniques.
Materials and Methods: Biodegradable polymeric microneedle arrays were fabricated out of poly lactic-co-glycolic acid (PLGA) using the micromolding technique under aseptic conditions, and the morphology of the microneedles was characterized using light microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to rule out drug-polymer interactions. Standard procedures were used to analyze the prepared microneedle arrays for in vitro drug release and to perform a microneedle insertion test. Enzyme-linked immunosorbent assay was used to quantify rHuKGF.
Results: The PLGA polymer was safe for use in the fabrication of rHuKGF microneedles as there was no interaction between the drug and the polymer. The fabricated rHuKGF microneedle arrays had fully formed microneedles with a height of 600 µm and a base of 300 µm. The drug from the microneedle patch was released in vitro within 30 minutes. The strength of the microneedles in the patch was good, as they were able to reach a depth of 381±3.56 µm into parafilm without any structural change or fracture.
Conclusion: Microneedle transdermal patches were successfully prepared for rHuKGF, and their evaluation suggested excellent quality and uniformity of patch characteristics. This can have potential applications in the therapeutic arena, offering advantages in terms of reduced dosing frequency, improved patient compliance, and bioavailability.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Detection of immunoglobulins M and G using culture filtrate antigen of Burkholderia pseudomallei

Chenthamarakshan V, Vadivelu J, Puthucheary SD

Diagn Microbiol Infect Dis, 2001 Jan;39(1):1-7.
PMID: 11173184

IgM and IgG based ELISA systems were developed using the culture filtrate antigen (CFA) of Burkholderia pseudomallei. The assays were evaluated using 95 sera from 66 septicemic cases and 47 sera from 20 cases with localized melioidosis. In addition 65 sera from culture negative cases that were also serologically negative for other endemic infections clinically suspected of melioidosis were included. These were compared with sera from 260 non-melioidosis cases, 169 sera from individuals with high risk of acquiring the infection and 48 sera from healthy controls. The IgG-ELISA was 96% sensitive and 94% specific. All sera from cases with septicemic and localized infections and 61 of 63 sera from clinically suspected melioidosis cases were positive for IgG antibody. The geometric mean titre index (GMTI) values of IgG antibody in melioidosis cases were significantly higher (p < 0.0005) compared to that of healthy subjects, high risk group and subjects with non-melioidosis infections. The sensitivity and specificity of IgM ELISA was 74 and 99% respectively. The GMTI value of IgM antibody in the sera of melioidosis cases was significantly higher as compared to that of non-melioidosis disease controls (p < or = 0.001). These results demonstrate that the detection of IgG is a better indicator of the disease in the diagnosis of melioidosis.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Isolation of human surfactant protein A from amniotic fluid

Cheong KB, Cheong SK, Boo NY, Jemilah M, Ton SH

Malays J Pathol, 1995 Dec;17(2):97-101.
PMID: 8935134

Surfactant protein A (SP-A) is one of the four known surfactant-associated proteins found in human lungs. It plays a major role in determining regulation of surfactant uptake and resecretion. Qualitative and quantitative deficiencies of SP-A may contribute to neonatal respiratory distress syndrome. The measurement of its level in amniotic fluid or neonatal tracheal aspirate may be useful in the assessment of replacement therapy using natural or synthetic surfactants. In order to develop an in-house immunoassay to detect the level of SP-A, we used a discontinuous sucrose density gradient to isolate SP-A from amniotic fluid. Polyacrylamide gel electrophoresis was carried out on the isolates with low molecular weight markers. We successfully isolated SP-A from 12 out of 31 samples of amniotic fluid. The isolates were found to be relatively pure and have a molecular weight of about 35 kD. The isolated SP-A were used as immunogens to raise antibodies in rabbits for the immunoassay.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Statistical optimization of green fluorescent protein production from Escherichia coli BL21(DE3)

Chew FN, Tan WS, Boo HC, Tey BT

Prep Biochem Biotechnol, 2012;42(6):535-50.
PMID: 23030465 DOI: 10.1080/10826068.2012.660903

An optimized cultivation condition is needed to maximize the functional green fluorescent protein (GFP) production. Six process variables (agitation rate, temperature, initial medium pH, concentration of inducer, time of induction, and inoculum density) were screened using the fractional factorial design. Three variables (agitation rate, temperature, and time of induction) exerted significant effects on functional GFP production in E. coli shake flask cultivation and were optimized subsequently using the Box-Behnken design. An agitation rate of 206 rpm at 31°C and induction of the protein expression when the cell density (OD(600nm)) reaches 1.04 could enhance the yield of functional GFP production from 0.025 g/L to 0.241 g/L, which is about ninefold higher than the unoptimized conditions. Unoptimized cultivation conditions resulted in protein aggregation and hence reduced the quantity of functional GFP. The model and regression equation based on the shake flask cultivation could be applied to a 2-L bioreactor for maximum functional GFP production.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Fluorescent quantitation method for differentiating the nativity of green fluorescent protein

Chew FN, Tan WS, Tey BT

J Biosci Bioeng, 2011 Feb;111(2):246-8.
PMID: 21036662 DOI: 10.1016/j.jbiosc.2010.10.004

A gel imaging method was employed to quantitate the GFP that had been subjected to denaturation and degradation treatments. This method is able to differentiate the nativity of GFP by relating the observed changes in the position of fluorescent bands which is unable to be detected using the spectrofluorometric method.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Quantitation of green fluorescent protein using a gel-based imaging method

Chew FN, Tan WS, Ling TC, Tan CS, Tey BT

Anal Biochem, 2009 Jan 15;384(2):353-5.
PMID: 18952038 DOI: 10.1016/j.ab.2008.10.010

Green fluorescent protein (GFP) is a versatile reporter protein and has been widely used in biological research. However, its quantitation requires expensive equipment such as a spectrofluorometer. In the current study, a gel documentation imaging system using a native polyacrylamide gel for the quantitation of GFP was developed. The assay was evaluated for its precision, linearity, reproducibility, and sensitivity in the presence of Escherichia coli cells and was compared with the spectrofluorometric method. Using this newly established, gel-based imaging technique; the amount of GFP can be quantified accurately.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel/methods*
Fulltext Single-step purification of the recombinant green fluorescent protein from intact Escherichia coli cells using preparative PAGE

Chew FN, Tan WS, Ling TC, Tey BT

Electrophoresis, 2009 Sep;30(17):3017-3023.
PMID: 19685471 DOI: 10.1002/elps.200900246

Mechanical and non-mechanical breakages of bacterial cells are usually the preliminary steps in intracellular protein purification. In this study, the recombinant green fluorescent protein (GFP) was purified from intact Escherichia coli cells using preparative PAGE. In this purification process, cells disruption step is not needed. The cellular content of E. coli was drifted out electrically from cells and the negatively charged GFP was further electroeluted from polyacrylamide gel column. SEM investigation of the electrophoresed cells revealed substantial structural damage at the cellular level. This integrated purification technique has successfully recovered the intracellular GFP with a yield of 82% and purity of 95%.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel/methods*
Fulltext Detection of glycoproteins from human erythrocytes of different ABO blood groups infected with Plasmodium falciparum

Chin, Ramon Beng Ong, Kim, Patricia Chooi Lim, Joon, Wah Mak

International e-Journal of Science, Medicine & Education, 2011;5(2):18-28.
MyJurnal

Background: Many proteins released by cells to the blood and other fluids are glycoproteins. One set of glycoproteins carry the ABO blood group determinants and glycoproteins have been shown to be vital in determining the structure and organization of plasma membranes. There is evidence suggesting their important role in cell-to-cell contact, adhesion, hormone interaction and vital transformation. Differences in proteins and glycoproteins in the different human blood groups may influence the invasion process of Plasmodium falciparum. The objectives of the study were to determine whether there are any changes in proteins and glycoproteins of red blood cells upon infection by P. falciparum and whether these protein and glycoprotein changes differ in the various ABO blood groups.

Methods: A Malaysian strain of P. falciparum was cultured in vitro in red blood cells from A, B, O and AB blood groups. Protein and glycoprotein profiles of uninfected and P. falciparum- infected red blood cells from the different human ABO blood groups were analyzed by SDS-PAGE. For protein bands, the gels were stained with Coomassie blue while glycoproteins were visualized following staining of gels using GelCode ® Glycoprotein Staining Kit.

Results: Cell membranes of P. falciparum infected erythrocytes from different ABO blood groups have different glycoprotein profiles compared to uninfected cells. All the infected samples showed a prominent protein band of molecular weight 99 kDa which was not present in any of the uninfected samples while a 48 kDa band was seen in four out of the seven infected samples. The erythrocyte cell membranes of A and AB blood groups showed different glycoprotein profiles upon infection with P. falciparum when compared to those from blood groups B and O.

Conclusion: The two glycoproteins of molecular weights 99 kDa and 48 kDa should be further studied to determine their roles in the pathogenesis of malaria and as potential targets for drug and vaccine development.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Fulltext Denaturing gradient-gel electrophoresis screening of familial defective apolipoprotein B-100 in a mixed Asian cohort: two cases of arginine3500-->tryptophan mutation associated with a unique haplotype

Choong ML, Koay ES, Khoo KL, Khaw MC, Sethi SK

Clin Chem, 1997 Jun;43(6 Pt 1):916-23.
PMID: 9191540

The Arg-to-Trp substitution at codon 3500 in the apolipoprotein (apo) B-100 gene is established as a cause of familial defective apo B-100 (FDB), a functional mutation, resulting in reduced LDL receptor binding and manifest hypercholesterolemia. In a search for similar mutations in 163 Malaysians, we screened the putative receptor-binding region (codons 3456-3553) of the apo B-100 gene by PCR amplification and denaturing gradient-gel electrophoresis. Four single-base mutations were detected and confirmed by DNA sequencing. Two females, a Chinese and a Malay, had the same CGG3500-->TGG mutation, resulting in an Arg3500-to-Trp substitution. This is the second published report of such an independent mutation involving the same codon as the established Arg3500-to-Gln mutation. The two other mutations detected, CTT3517-->CTG and GCC3527-->GCT, resulted in degenerate codons with no amino acid substitutions. All four mutations were associated with a unique apo B haplotype, different from those found in Caucasian FDB patients but concurring with that previously reported for two other Asians with FDB.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel/methods*
Plant proteins, minerals and trace elements of Eurycoma longifolia (Tongkat Ali)

Chua LS, Abdul-Rahman N, Rosidi B, Lee CT

Nat Prod Res, 2013 Mar;27(4-5):314-8.
PMID: 22468741 DOI: 10.1080/14786419.2012.676552

A water extraction method has been used to extract plant proteins from the roots of Eurycoma longifolia harvested from Perak and Pahang, Malaysia. On the basis of the spectroscopic Bradford assay, Tongkat Ali Perak and Pahang contained 0.3868 and 0.9573 mg mL(-1) of crude protein, respectively. The crude proteins were separated by one dimensional 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis into two (49.8 and 5.5 kD) and four (49.8, 24.7, 21.1 and 5.5 kD) protein spots for Tongkat Ali Perak and Pahang, respectively. Isoleucine was present in the highest concentration significantly. Both plant samples showed differences in the mineral and trace element profiles, but the minerals calcium, magnesium and potassium were present in the highest concentration. The highly concerned toxic metals such as arsenic and lead were not detected.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
The rubber elongation factor of rubber trees (Hevea brasiliensis) is the major allergen in latex

Czuppon AB, Chen Z, Rennert S, Engelke T, Meyer HE, Heber M, et al.

J Allergy Clin Immunol, 1993 Nov;92(5):690-7.
PMID: 8227860

BACKGROUND: Allergy to latex-containing articles is becoming more and more important because it can result in unexpected life-threatening anaphylactic reactions in sensitized individuals.
METHODS: A protein of 58 kd with an isoelectric point of 8.45 was purified from raw latex and from latex gloves and identified as the major allergen, completely blocking specific IgE antibodies in the serum of latex-sensitized subjects. The allergen is a noncovalent homotetramer molecule, in which the 14.6 kd monomer was identified, by amino acid composition and sequence homologies of tryptic peptides, to be the rubber elongation factor found in natural latex of the Malaysian rubber tree.
RESULTS: Competitive immunoinhibition tests showed that the starch powder covering the finished gloves is the airborne carrier of the allergen, resulting in bronchial asthma on inhalation. The purified allergen can induce allergic reactions in the nanogram range.
CONCLUSION: The identification of the allergen (Hev b I) may help to eliminate it during the production of latex-based articles in the future.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Electrophoretic profiles and biological activities: intraspecific variation in the venom of the Malayan pit viper (Calloselasma rhodostoma)

Daltry JC, Ponnudurai G, Shin CK, Tan NH, Thorpe RS, Wüster W

Toxicon, 1996 Jan;34(1):67-79.
PMID: 8835335

The Malayan pit viper (Calloselasma rhodostoma) is of major clinical significance both as a leading cause of snakebite and as the source of ancrod (Arvin). Although its venom has been extensively studied, the degree to which venom composition varies between individuals is poorly known. We individually analysed the venoms of over 100 C. rhodostoma using isoelectric focusing. In all populations, females produced an intense band that was absent from all males, and significant ontogenetic variation was detected. Principal components analysis of the banding profiles also revealed strong geographic variation, which was significantly congruent with variation in the biological activities of the venom (phosphodiesterase, alkalinephosphoesterase, L-amino acid oxidase, arginine ester hydrolase, 5'-nucleotidase, thrombin-like enzyme, haemorrhagic activity). Studies of captive-bred snakes indicate that the intraspecific variation in venom is genetically inherited rather than environmentally induced. The intraspecific variation in venom composition and biological activity could be of applied importance to snakebite therapy, both in correct diagnosis of the source of envenomation and in the development of a more effective antivenom. Greater attention should be given to the source of C. rhodostoma venom used in research to ensure reproducibility of results.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Site-Directed Mutagenesis of Cytochrome P450 2D6 and 2C19 Enzymes: Expression and Spectral Characterization of Naturally Occurring Allelic Variants

Dong AN, Pan Y, Palanisamy UD, Yiap BC, Ahemad N, Ong CE

Appl Biochem Biotechnol, 2018 Sep;186(1):132-144.
PMID: 29524040 DOI: 10.1007/s12010-018-2728-0

Genetic polymorphism of the cytochrome P450 (CYP) genes particularly affects CYP2D6 and CYP2C19 to a functionally relevant extent, and it is therefore crucial to elucidate the enzyme kinetic and molecular basis for altered catalytic activity of these allelic variants. This study explored the expression and function of the reported alleles CYP2D6*2, CYP2D6*10, CYP2D6*17, CYP2C19*23, CYP2C19*24, and CYP2C19*25 with respect to gene polymorphisms. Site-directed mutagenesis (SDM) was carried out to generate these six alleles. After DNA sequencing, the CYP2D6 and CYP2C19 wild types alongside with their alleles were each independently co-expressed with NADPH-CYP oxidoreductase (OxR) in Escherichia coli. The expressed proteins were analyzed using Western blotting, reduced carbon monoxide (CO) difference spectral scanning, and cytochrome c reductase assay. Results from Western blot revealed the presence of all CYP wild-type and allelic proteins in E. coli membrane fractions. The reduced CO difference spectra scanning presented the distinct peak of absorbance at 450 nm, and the cytochrome c reductase assay has confirmed that spectrally active OxR was expressed in each protein preparation. As a conclusion, the results obtained from this study have proven the CYP variants to be immunoreactive and spectrally active and are suitable for use to examine biotransformation and interaction mechanism of the enzymes.

Matched MeSH terms: Electrophoresis, Polyacrylamide Gel

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