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The incidences of methicillin-resistant strains of Staphylococcus aureus (MRSA) and their survival inside the macrophages are the major attributes of the relapsed infections after antimicrobial therapy, and it is a global problem. In this context, we have previously demonstrated 4-methoxy-1-methyl-2-oxopyridine-3-carbamide (MMOXC), a Ricinine derivative exhibiting anti-S. aureus and anti-biofilm characteristics by competitively inhibiting uridine monophosphate kinase (UMPK), UDP-N-acetyl muramyl pentapeptide ligase (Mur-F), and peptidyl deformylase, (PDF). In the present study, the stability of this competitive inhibitor MMOXC was evaluated by showing its ability to remain bound to the active sites of UMPK, Mur-F, and PDF even after increasing the incubation time, temperature, pH, and substrate concentration. On growing MRSA in fewer concentrations of MMOXC, these strains could not attain resistance to MMOXC and at the same time distinct reductions in the expression of UMPK, Mur-F, and PDF genes were noted. In vitro, infective models were generated by infecting MRSA to RAW 264.7 and human monocyte-derived macrophage (hMDM) cell lines. In these infected cell lines, in spite of increased nitric oxide synthase (NOS), NADPH-P450 reductase, superoxide dismutase, catalase, and peroxidase activities, the MRSA survived. At 640 µM/ml, the concentration of MMOXC penetrated into these infected cells and obliterated MRSA. While treating uninfected macrophage cell lines with MMOXC, no appreciable effect was observed indicating that MMOXC is the most suitable drug for the treatment of infections caused by MRSA.

The enormous spread of Staphylococcus aureus infections through biofilms is a major concern in hospital-acquired infections. Biofilm formation by S. aureus on any surface is facilitated by adjusting its redox status. This organism is a facultative anaerobe shift more towards reductive conditions by enhancing nitrogen metabolism where glutamine synthesis plays a key role. Glutamine is synthesized by glutamine synthetase (GS) encoded by the glnA gene. The gene was amplified by PCR from the chromosomal DNA of S. aureus, sequenced (HQ329146.1), and cloned. The pure recombinant GS exhibited Km of 11.06 ± 0.05 mmol·L−1 for glutamate and 2.4 ± 0.03 mmol·L−1 for ATP. The glnA gene sequence showed a high degree of variability with its human counterpart, while it was highly conserved in bacteria. Structural analysis revealed that the GS structure of S. aureus showed close homology with other Gram-positive bacteria and exhibited a high degree of variation with Escherichia coli GS. In the present study, we observed the increased presence of GS activity in multidrug-resistant strains of S. aureus with elevated biofilm units, grown in brain heart infusion broth; among them methicillin-resistant strains S. aureus LMV 3, 4, and 5 showed higher biofilm units. All these results explain the important role of glutamine biosynthesis with elevated biofilm units in the pathogenesis of S. aureus.

Congenital Erythrocytosis (CE) can be primary or secondary due to the mutations in genes involved in the erythropoietin receptor and oxygen sensing pathway. In this study, 42 patients with 38 unrelated patients and one family (4 patients) who were JAK-2 mutation (both exon 12 and exon 14) negative with high haematocrit values were investigated. The Endogenous Erythroid colony (EEC) assay was performed in all patients, interestingly EEC colonies were high in EPAS1 and EPOR mutated patients compared to non-mutated patients. The sequence analysis of EPAS1 (exon 12), EPO-R (exon-8), VHL (exon-3), and EGLN1 (exon-1) genes in all these patients showed 19% of patients (8/42) had mutations, in exon12 of EPAS1 and exon 8 of EPO-R genes. Two novel missense mutations MW_600850:c.1183GC, MW_600851:c.1028AC in EPO-R gene were observed in the study group. One new MW_600849:c.1969CT nonsense mutation and five MW_619914:c.1715AG, MW_619915:c.1694GT, MW_619916:c.1634TC, MW_600852:c.1771CG, MW_600848:c.1859GA novel missense mutations were observed in the EPAS1 gene. Among them, 4 mutations p. (Gln572Arg), p. (Ser565Ile), p. (Ile545Thr), p. (Gln591Glu) in the ODD (Oxygen-dependent degradation) domain of HIF2α, all these variations contributed to the formation of non-functional HIF2α. No mutations were observed in VHL and EGLN1 genes. Using in silico analysis we observed that these mutations contributed to major conformational changes in the HIF2α protein making it non-functional. The mutations in the EPAS1 gene were heterozygous and show autosomal dominant inheritance patterns and we observed in one family. These novel mutations in the EPAS1 (75% (6/8)) and 25% (2/8) EPO-R genes correlating with EEC positivity were observed for the first time in India in CE patients.

Haematopoietic stem cells (HSCs) possess multipotent ability to differentiate into various types of cells on providing appropriate niche. In the present study, the differentiating potential of human HSCs into β-cells of islets of langerhans was explored. Human HSCs were apheretically isolated from a donor and cultured. Phenotypic characterization of CD34 glycoprotein in the growing monolayer HSCs was confirmed by immunocytochemistry and flow cytometry techniques. HSCs were induced by selection with beta cell differentiating medium (BDM), which consists of epidermal growth factor (EGF), fibroblast growth factor (FGF), transferrin, Triiodo-l-Tyronine, nicotinamide and activin A. Distinct morphological changes of differentiated cells were observed on staining with dithizone (DTZ) and expression of PDX1, insulin and synaptophysin was confirmed by immunocytochemistry. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed distinct expression of specific β-cell markers, pancreatic and duodenal homeobox-1 (PDX1), glucose transporter-2 (GLUT-2), synaptophysin (SYP) and insulin (INS) in these differentiated cells compared to HSCs. Further, these cells exhibited elevated expression of INS gene at 10 mM glucose upon inducing with different glucose concentrations. The prominent feature of the obtained β-cells was the presence of glucose sensors, which was determined by glucokinase activity and high glucokinase activity compared with CD34(+) stem cells. These findings illustrate the differentiation of CD34(+) HSCs into β-cells of islets of langerhans.

Osteocytic potentiality of human CD34+ stem cells explored in the present study by generating in vitro agarose gel 3D model to understand the bone ossification process. The G-CSF and IL-3 mobilized human CD34+ stem cells isolated apheretically from donor peripheral blood and purity of the cells was assessed by FACS and immunocytochemical (ICC) studies. The CD34+ stem cells were cultured in gel based 3D model with osteogenic stimulating medium for 21 days. The transition stages from undifferentiated to differentiated osteocytes through osteoblasts were studied with expression markers Differentiated cells at Day 7 showed positive reactivity with monoclonal anti-Runx2, an early osteoblastic marker. qPCR expression analysis showed early and mature osteoblastic markers like RUNX2, Osterix, RANKL, along with osteocyte markers SPARC, Sclerostin. While poor expression of OSCAR genes was observed apart from conspicuous expression of alkaline phosphatase. The expression of sclerostin and SPARC suggests that these differentiated cells are behaving like true osteocytes, sclerostin expression causes transformation of osteoblast into osteocytes and negligible expression of OSCAR, RANK, NFATc and cathepsin K genes explains there are no osteoclasts in the differentiated culture. These cells showed positive reaction with Alizarin red stain indicating expression of calcium bound bone morphogenic proteins like osteonectin. All these results clearly confirm the human CD34+ stem cells possess unique osteogenic differentiation potential and can be used in the early regeneration of injured bone.

Pyogenic Arthritis, Pyoderma gangrenosum, and Acne (PAPA syndrome) is a rare autosomal dominant, auto-inflammatory disease that affects joints and skin. The disease results due to mutations in the cluster of differentiation 2 binding protein 1 (CD2BP1) gene on chromosome 15q24.3. Rheumatoid arthritis (RA) is a common, genetically complex disease that affects the joints with occasional skin manifestations. Studies related to the pathophysiology of inflammation in these two disorders show a certain degree of overlap at genetic level. The present study was done to confirm the existence of such a genetic overlap between PAPA syndrome and RA in south Indian population. In the present study 100 patients who were clinically diagnosed rheumatoid arthritis and 100 apparently healthy controls were chosen and the 15 exons of CD2BP1 gene were PCR-amplified and sequenced. The sequence analysis showed that in exon 3 thirty eight patients revealed presence of novel heterozygous missense mutations p.Glu51Asp, p.Leu57Arg and p.Ala64Thr. In exons 6, 10 and 14 eight patients showed 44 novel missense mutations and two patients showed novel frame shift mutations p.(Met123_Leu416delinsThr) and p.(Thr337Profs*52) leading to truncated protein formation. Such mutations were not seen in controls. Further, the in silico analysis revealed the mutant CD2BP1 structure showed deletion of Cdc15 and SH3 domains when superimposed with the wild type CD2BP1 structure with variable RMSD values. Therefore, these structural variations in CD2BP1 gene due to the mutations could be one of the strongest reasons to demonstrate the involvement of these gene variations in the patients with rheumatoid arthritis.

Isocitrate dehydrogenase (IDH) gene from Staphylococcus aureus ATCC12600 was cloned, sequenced and characterized (HM067707). PknB site was observed in the active site of IDH; thus, it was predicted as IDH may be regulated by phosphorylation. Therefore, in this study, PknB, alkaline phosphatase III (SAOV 2675) and IDH genes (JN695616, JN645811 and HM067707) of S. aureus ATCC12600 were over expressed from clones PV 1, UVPALP-3 and UVIDH 1. On passing the cytosloic fractions through nickel metal chelate column, pure enzymes were obtained. Phosphorylation of pure IDH by PknB resulted in the complete loss of activity and was restored upon dephosphorylation with SAOV 2675 which indicated that phosphorylation and dephosphorylation regulate IDH activity in S. aureus. Further, when S. aureus ATCC12600 was grown in BHI broth, decreased IDH activity and increased biofilm units were observed; therefore, this regulation of IDH alters redox status in this pathogen favouring biofilm formation.

Neurofibromatosis type 1 (NF1) is a rare autosomal-dominant disorder caused by inactivation of NF1 tumour suppressor gene, which associates in the development of peripheral nerve tumours. NF1 is an important regulator of GAP and RAS proteins, mutations in NF1 results in the impairment in this function causing specific osseous lesions in any organ of the human body. In the present study, we investigated the clinical characteristics and NF1 gene mutation analysis of 3 unrelated Indian families with neurofibromatosis type 1. All the exons of NF1 gene was PCR amplified and sequenced. The structural and functional analysis was performed using molecular modelling tools. The sequence analysis of NF1 gene revealed; in family I five novel mutations p.R103K, p.D105N, p.M108I, p.L114M, p.E116X and p.A131S was observed in exon 4. In family II one missense p.A131S mutation and one silent p.L234L mutation was detected in exon 4. While, in family III one novel frame shift p.E225Rfs∗6 mutation was identified in exon 7 resulting in the truncated protein formation. Further, the structural analysis revealed all these mutations fall in the protein kinase C domain of NF1 gene causing loss of functional GRD and CSRD domains. In conclusion, novel mutations in the exon 4 and exon 7 of NF1 gene in these families correlating with genotype-phenotype characters explaining the neurofibromatosis type 1 and peripheral nerve sheath tumours condition in these patients.

The failure of regeneration of damaged liver in various end-stage liver diseases results in high morbidity and mortality. In this context, we have demonstrated the differentiation ability of human hematopoietic stem cells (HSCs) into hepatocytes. In this study, HSCs were isolated from a donor and cultured which exhibited the presence of CD34 and CD133 and absence of CD90 and CD73 markers. These CD34+ HSCs were induced for 21 days in hepatocyte differentiation medium (HDM). The obtained cells were characterized by immunocytochemical, immunofluorescence, western blot, qRT-PCR and flow cytometry analysis. Further, functional assays were done to accentuate the differentiated cells are hepatocytes. In HDM at day 6 differentiated cells showed the expression of definitive endodermal (DE) markers, SOX17, GATA4 and FoxA2 indicating the beginning of differentiation process. At day 21 the flow cytometry analysis showed 84.2 % positive to ALB-PE, 75.4 % positive to HNF4α-PE, and 77.3% positive to AFP-PE. Further, the qRT-PCR and western blot analysis presented prominent expression of hepatocyte-specific genes AAT, ALB, AFP, CK18, CK19, HNF4α, TFR2, and Hepcidin confirms the generation of hepatocytes in HDM. The ability of albumin secretion, urea production, glycogen storage, uptake of LDL, high ALDH enzyme activity describes the functionality of differentiated hepatocytes. Distinct expression of UGT1A1, CYP2B6, CYP2C9, CYP3A4, and CYP7A1 genes explains the ability to clear toxins and bilirubin as observed in normal hepatocytes. All these results indicate HSCs were differentiated into hepatocytes thus, autologous transplantation of HSCs could be a better option in the regeneration of the damaged liver.

Staphylococcus aureus a natural inhabitant of nasopharyngeal tract mainly survives as biofilms and possess complete Krebs cycle which plays major role in its pathogenesis. This TCA cycle is regulated by Isocitrate dehydrogenase (IDH) we have earlier cloned, sequenced (HM067707), expressed and characterized this enzyme from S. aureus ATCC12600. We have observed only one type of IDH in all the strains of S. aureus which dictates the flow of carbon thereby controlling the virulence and biofilm formation, this phenomenon is variable among bacteria. Therefore in the present study comparative structural and functional analysis of IDH was undertaken. As the crystal structure of S. aureus IDH was not available therefore using the deduced amino sequence of complete gene the 3D structure of IDH was built in Modeller 9v8. The PROCHECK and ProSAweb analysis showed the built structure was close to the crystal structure of Bacillus subtilis. This structure when superimposed with other bacterial IDH structures exhibited extensive structural variations as evidenced from the RMSD values correlating with extensive sequential variations. Only 24% sequence identity was observed with both human NADP dependent IDHs (PDB: 1T09 and 1T0L) and the structural comparative studies indicated extensive structural variations with an RMSD values of 14.284A and 10.073A respectively. Docking of isocitrate to both human IDHs and S. aureus IDH structures showed docking scores of -11.6169 and -10.973 respectively clearly indicating higher binding affinity of isocitrate to human IDH.

It has been observed that mycobacterial species has high content of cardiolipin (CL) in their cell membranes more so pathogenic mycobacteria and in bacteria CL activates polymerases, gyrases by removing the bound ADP. Therefore, in the present study cardiolipin synthase (cls) which catalyses the formation of CL was isolated purified and characterized from the cell membrane of Mycobacterium phlei. The purified cls obtained from C-18 RP-HPLC column had a molecular weight of 58 kDa with an isoelectric point of 4.5. The enzyme activity (11.5+0.15 µM of CL phosphorous. ml-1 minute-1 for PG as substrate and 14+0.35µM of CL phosphorous. ml-1 minute-1 for CDP-DG as substrate) was optimal at pH 4.8 and showed KM values of 55+0.05µM and 2.56+0.04µM for phosphatidyl glycerol and CDP-diacylglycerol, respectively, with an absolute requirement of Mg(2+) and Mn(2+) ions for its activity however, Ca(2+) ions inhibited the activity of the cls. The partial amino acid sequence of cls showed significant homology with pgsA3 gene of M. tuberculosis and in this organism the CL biosynthesis is very high having three genes coding for PLs biosynthesis therefore, enzymes involved in CL biosynthesis may be an attractive drug target in the development of new antimycobacterial drugs.

In Staphylococcus aureus, adherence and secretory proteins play chief role in the formation of biofilms. This mode of growth exhibits resistance to a variety of antibiotics and spreads its infections. In the present study, secretary and adherence proteins, Protein-A, Fibronectin-binding protein-A (FnbA) and Rsp (a transcription regulator encoding proteolytic property) expression levels were evaluated at different stages of growth in S. aureus ATCC12600 a drug-sensitive strain and multidrug-resistant strains of S. aureus. Initially, the SpA, FnbA and Rsp genes of S. aureus ATCC12600 were cloned, sequenced, expressed and characterized. The proteolytic property of recombinant Rsp was conspicuously shown when this pathogen was grown in aerobic conditions correlating with reduced biofilm units. In anaerobic mode of growth, S. aureus exhibited a higher expression of SpA and FnbA in early and mid adherence phases and finally stabilized at 48 h of incubation. This expression was more pronounced in methicillin-resistant strains (LMV1-8 and D1-4) of S. aureus. In all these stages, Rsp gene expression was at the lowest level and these results concur with the increased biofilm units. The results of the present study explain proteins chiefly contribute in the formation of biofilms.

Autosomal recessive distal renal tubular acidosis (dRTA) is associated with mutation in the ATP6B1 gene encoding the B1 subunit of H + -ATPase, one of the key membrane transporters for net acid excretion of α-intercalated cells of medullary collecting ducts. Sensori-neural deafness frequently accompanies this type of dRTA. We herewith describe a patient who had distinct features of dRTA with bilateral sensori-neural hearing loss and ATP6B1 mutation. This is a rare entity.

Human CD34+ stem cells differentiated into type-II pneumocytes in Dulbecco’s modified Eagle medium (DMEM) having hydrocortisone, insulin, fibroblast growth factor (FGF), epidermal growth factor (EGF) and bovine serum albumin (BSA), expressing surfactant proteins-B (SP-B) and C (SP-C), alkaline phosphatase (ALP) and lysozyme. FACS-enumerated pure CD34+ cells, isolated from human peripheral blood, were cultured in DMEM and showed positive reaction with anti-human CD34 monoclonal antibodies in immunocytochemistry. These cells were cultured in DMEM having hydrocortisone, insulin, FGF, EGF and BSA (HIFEB-D) medium having an air–liquid interface. They differentiated into type-II pneumocytes with expression of SP-B and SP-C genes and disappearance of CD34 expression as assessed using real-time PCR. In reverse transcription-PCR amplicons showed 208 and 907 bp confirming SP-B and SP-C expressions. These cells expressed ALP with an activity of 1.05 ± 0.09 mM ml−1 min−1 and lysozyme that killed E. coli. The successful differentiation of human CD34+ stem cells into type-II pneumocytes, and transplantation of such cells obtained from the patient’s stem cell could be the futuristic approach to regenerate diseased lung alveoli.

Distal renal tubular acidosis (dRTA) is an autosomal recessive syndrome results defect in either proximal tubule bicarbonate reabsorption or in distal tubule H(+) secretion and is characterized by severe hyperchloraemic metabolic acidosis in childhood. dRTA is associated with functional variations in the ATP6V1B1 gene encoding β1 subunit of H(+)-ATPase, key membrane transporters for net acid excretion of α-intercalated cells of medullary collecting ducts. In the present study, a 13-year-old male patient suffering with nephropathy and sensorineural deafness was reported in the Department of Nephrology. We predicted improper functioning of ATP6V1B1 gene could be the reason for diseased condition. Therefore, exons 3, 4, and 7 contributing active site of ATP6V1B1 gene was amplified and sequenced (Accession numbers: KF571726, KM222653). The obtained sequences were BLAST searched against the wild type ATP6V1B1 gene which showed novel mutations c.307 A G, c.308 C A, c.310 C G, c.704 T C, c.705 G T, c.709 A G, c.710 A G, c.714 G A, c.716 C A, c.717delC, c.722 C G, c.728insG, c.741insT, c.753G C. These mutations resulted in the expression of truncated protein terminating at Lys 209. The mutated ATP6V1B1structure superimposed with wild type showed extensive variations with RMSD 1.336 Å and could not bind to substrate ADP leading to non-functional ATPase. These results conclusively explain these mutations in ATP6V1B1 gene resulted in structural changes causing accumulation of H(+) ions contributing to dRTA with sensorineural deafness.

Mutations in the glucokinase (GK) gene play a critical role in the establishment of type 2 diabetes. In our earlier study, R308K mutation in GK in a clinically proven type 2 diabetic patient showed, structural and functional variations that contributed immensely to the hyperglycemic condition. In the extension of this work, a cohort of 30 patients with established type 2 diabetic condition were chosen and the exons 10 and 11 of GK were PCR-amplified and sequenced. The sequence alignment showed A379S, D400Y, E300A, E395A, E395G, H380N, I348N, L301M, M298I, M381G, M402R, R308K, R394P, R397S, and S398R mutations in 12 different patients. The structural analysis of these mutated GKs, showed a variable number of β-α-β units, hairpins, β-bulges, strands, helices, helix–helix interactions, β-turns, and γ-turns along with the RMSD variations when compared to wild-type GK. Molecular modeling studies revealed that the substrate showed variable binding orientations and could not fit into the active site of these mutated structures; moreover, it was expelled out of the conformations. Therefore, these structural variations in GK due to mutations could be one of the strongest reasons for the hyperglycemic levels in these type 2 diabetic patients.

Aspergillus fumigatus is an opportunistic fungus which causes pulmonary complications in humans and animals. The clinical spectrum observed with A. fumigatus is attributed to the multifunctional nature of its antigens. Lack of understanding on the molecular processes and complexity of the fungus have spurred interest in the identification and characterization of its antigens/allergens with biological activities and virulence functions. For identification of some of these antigens/allergens, a cDNA library of A. fumigatus was screened with antibodies of allergic bronchopulmonary aspergillosis (ABPA) patients. One of the reactive clones was sequenced and observed to have an open reading frame of 1095 nucleotides corresponding to a polypeptide of 364 amino acids. The nucleotide and deduced amino acid sequence showed significant homology with the protein disulfide isomerase (PDI) superfamily. The expressed recombinant fusion protein exhibited specific IgG and IgE binding with antibodies present in ABPA patients' sera. The recombinant protein in vitro catalyzed folding of scrambled RNase. The probable epitopic regions of the deduced amino acid sequence were mapped by algorithmic analysis. This is the first report of isolation of a gene encoding a member of the PDI family from A. fumigatus. The PDI superfamily of proteins may play an important role in the protein folding mechanisms of A. fumigatus antigens/allergens for their interaction with the host.

Staphylococcus aureus is one of the prominent Gram positive human pathogen secretes many surface and secretary proteins including various enzymes and pathogenic factors that favour the successful colonization and infection of host tissue. α-amylase is one of the enzymes secreted by S. aureus which catalyses the breakdown of complex sugars to monosaccharides, which are required for colonization and survival of this pathogen in any anatomical locales. In the present study we have cloned, sequenced, expressed and characterized α-amylase gene from S. aureus ATCC12600. The recombinant enzyme has a molecular weight of 58kDa and the kinetics showed Vmax 0.0208±0.033 (mg/ml)/mg/min and Km 10.633±0.737mg/ml. The multiple sequence analysis showed α- amylase of S. aureus exhibited large differences with Bacillus subtilis and Streptococcus bovis. As the crystal structure of S. aureus α- amylase was unavailable, we used homology modelling method to build the structure. The built structure was validated by Ramachandran plot which showed 90% of the residues in the allowed region while no residue was found in the disallowed region and the built structure was close to the crystal structure with Z-Score: -6.85. The structural superimposition studies with α- amylases of Bacillus subtilis and Streptococcus bovis showed distinct differences with RMSD values of 18.158Aand 7.091A respectively which correlated with enzyme kinetics, indicating α-amylase is different among these bacteria.

Glucose-6-phosphate (G-6-P) formation in Staphylococcus aureus is catalysed by glucokinase (glkA) gene under high glucose concentration leading to upregulation of various pathogenic factors; therefore the present study is aimed in the cloning and characterization of glk A gene from S. aureus ATCC12600. The glk A gene was cloned in the Sma I site of pQE 30, sequenced (Accession number: JN645812) and expressed in E. coli DH5α. The recombinant glk A expressed from the resultant glk A 1 clone was purified using nickel metal chelate chromatography, the pure enzyme gave single band in SDS-PAGE with molecular weight of 33kDa. The rglk A showed very high affinity to glucose Km 5.1±0.06mM with Hill coefficient of 1.66±0.032mM. Analysis of glucokinase sequence of S. aureus showed presence of typical ATP binding site and ROK motif CNCGRSGCIE. Sequentially and phylogenetically S. aureus glk A exhibited low identity with other bacterial glk A and 21% homology with human glucokinase (GCK). Functionally, S. aureus glk A showed higher rate of G-6-P formation compared to human GCK which may have profound role in the pathogenesis.

The Krebs cycle dictates oxidative and reductive conditions in Staphylococcus aureus and is mainly regulated by isocitrate dehydrogenase (IDH) which plays pivotal role in the growth and pathogenesis of the bacteria. In the present study, IDH gene from S. aureus ATCC12600 was cloned in the Sma I site of pQE 30 vector; the resultant clone was named as UVIDH1. The insert in the clone was sequenced (accession number HM067707), and the sequence showed complete homology with IDH sequence of other S. aureus strains reported in the database indicating presence of single enzyme in S. aureus, and considerable sequence homology with other bacteria was observed; however, only 24% homology was found with NADP-dependent human IDH. Phylogenetically, the S. aureus IDH showed close identity with Bacillus subtilis and high degree of variability with other bacteria and human IDH. The expression of IDH in the clone UVIDH1 was induced with 1 mM IPTG, and the recombinant IDH was purified by passing through nickel metal chelate column; the purified recombinant IDH showed a single band in SDS-PAGE with a molecular weight of 40 kDa; K(m) and V(max) for isocitrate are 8.2 ± 0.28 and 525 ± 25 μM NADPH/mg/min, respectively, and for cofactor NADP 67.5 ± 2.82 μM and V(max) 50.5 ± 2.12 μM NADPH/mg/min.