M. Waheed Akhtar

Proteomic identification of human serum biomarkers in diabetes mellitus type 2

Discovery of protein biomarkers in different diseases is an important area of research in the field of proteomics. We have described the levels of protein biomarkers specific to diabetes mellitus type 2 in the local population of Pakistan using proteomic technology. Type 2 diabetic patients, age and sex-matched normal healthy controls were recruited from Sheikh Zayed Hospital, Lahore, Pakistan. Plasma proteins were analysed by 2D liquid chromatographic system in which samples were initially fractionated by chromatofocusing and the selected fractions were further analysed by reverse-phase high performance liquid chromatography. The proteins which showed variation between test and control samples were identified by MALDI-TOF analysis. All the samples belonging to the control and diabetic groups were then analyzed by ELISA and estimated four proteins which were found to vary. Levels of apolipoprotein A-I was found to decrease by -6.4% while apolipoprotein E, leptin and C reactive protein (CRP) were increased by +802, +842 and +872%, respectively, in the diabetic patients as compared to the controls. The discovery of these marker proteins might thus provide an adjunctive method for early detection of risk for this disease.

Removal and recovery of zirconium from its aqueous solution by Candida tropicalis

Removal and recovery of zirconium from dilute aqueous solutions by Candida tropicalis used as biosorbent, was studied by performing biosorption-desorption tests. This biosorbent was selected after screening a range of microbial species. The process was found to be highly dependent on initial pH and concentration of metal solution. At optimized experimental parameters, the maximum zirconium biosorption capacity of C. tropicalis was 179 mg Zr g(-1) dry weight of biosorbent. The adsorption distribution coefficient value of 3968 ml g(-1) was obtained for zirconium biosorption by C. tropicalis. Different theoretical thermodynamic models governing the adsorption behavior of zirconium were also tested. Zirconium biosorption was found to closely follow the Langmuir model. At low biomass concentrations it was found to follow pseudo-first-order kinetics. However when higher biomass concentrations were used kinetics was changed to pseudo-second-order. The zirconium bound to the biomass was stripped out (60.2% at S/L of 1.0 g of zirconium loaded biomass/l of eluent) using sodium bicarbonate and the biomass could be used for multiple sorption-desorption cycles.

Production of free and substrate-bound cellulases of cellulomonas flavigena

Abstract Conditions for the production of extracellular carboxymethyl cellulase ( CMCase ) and avicelase activities by a locally isolated Cellulomonas species identified as C. flavigena were optimized. The microbe produced maximal levels of free cellulases after 72 h of fermentation, when cultivated in the presence of 0.2% yeast extract, 0.5% Avicel, and 0.1% Tween 80 at 30°C. Conditions for the elution of substrate-bound cellulases from the residual Avicel were optimized. Higher proportions of the extracellular cellulases were bound to the substrate when higher concentrations of Avicel were used. When 2.0% Avicel was used as a carbon source, maximal levels of extracellular CMCase and avicelase produced were 13.2 and 3 U ml −1 of the culture medium, respectively. Both the cellulases were most active at pH 6.5 and 50°C and were inhibited by β-mercaptoethanol. Calcium chloride activated CMCase but had no effect on avicelase activity. Each of the enzymes lost activity when incubated at 70°C for 30 min. A comparative study of free and substrate-bound CMCase on gradient polyacrylamide gel electrophoresis ( PAGE ) showed that free CMCase activity was composed of at least seven active fractions, while bound activity had four major bands of CMCase activity. Since a considerable level of protease was secreted by C. flavigena, the additional fractions in the free CMCase activity could be a result of proteolysis .

Purification and characterization of two low-molecular weight endoglucanases of Cellulomonas flavigena

Abstract Two isoforms of endoglucanases of Cellulomonas flavigena were purified to homogeneity after a series of purification steps including precipitation with 80% acetone, gel filtration, ion-exchange chromatography, and nondenaturing gradient 5–20% preparative polyacrylamide gel electrophoresis. Each of the endoglucanases, CM-Cellulase 1 and CM-Cellulase 2, appeared as a single band on SDS-PAGE and had an apparent molecular weight of 20,400. Both of the endoglucanases were comparable to the substrate-bound endoglucanases and the recombinant DNA-derived endoglucanases of C. flavigena expressed in Escherichia coli. The two isoforms of endoglucanase, CM-Cellulase 1 and CM-Cellulase 2, were different in their mobilities on native nondenaturing 5–20% gradient PAGE. The purified endoglucanases displayed significantly similar characteristics, including pH optima (6.5 and 7) and temperature optima (50°C). The purified CM-Cellulases were unable to hydrolyze Avicel, xylan, and filter paper and were only active against CMC. The purified proteins had a K m value of 0.78 g l −1 . Heavy metal ions like Ag 2+ and Fe 2+ inactivated the enzymes. NaCl and CaCl 2 had no effect on enzyme activities. β-Mercaptoethanol also inhibited enzyme activity, possibly due to the involvement of sulfhydryl groups .

Role of silent gene mutations in the expression of caprine growth hormone in Escherchia coli.

This report describes the strategy for overexpression of caprine growth hormone (cGH) gene of beetal goat in E. coli through introducing silent mutations in the 5′‐end of the coding sequence. The silent mutations introduced were aimed at minimizing translation‐inhibiting secondary structures in the mRNA. Free energies of the resultant mRNAs were calculated from the ribosomal binding site of mRNA to +24 base using the Mfold web server. The construct with native sequence did not show any expression, whereas introduction of the silent mutations had strong influence on the expression levels. Some constructs (pETcGH2–7) showed 12–30% expression of total cell proteins while some others (pETcGH8–16) showed 30 to 53% of total cell protein. Any variation in the amount of mRNA transcript for the various constructs, as determined by quantitative PCR, was not enough to suggest that the variable level of the gene expression was due to variation in the transcription levels. It appears that the expression levels are not always correlated with free‐energy values of the secondary structures in the 5′‐end region of the mRNA; instead some key silent nucleotide alterations at certain sites of 5′‐end of the sequence reorganize the secondary structure in such a way that it has positive impact on translation without considerably altering the free‐energy values. An empirical approach for determining the optimum 5′‐end substitutions for hyperexpression of a recombinant protein thus seems necessary.

Truncation of PstS1 antigen of Mycobacterium tuberculosis improves diagnostic efficiency

PstS1, also named 38-kDa antigen, is one of the earliest known immune-dominant antigens of Mycobacterium tuberculosis and it has been commonly used in serodiagnostic tests. We constructed a truncated version, tnPstS1, by removing 96 and 14 amino acid residues from the N- and C-terminals, respectively of the native PstS1. The native and the truncated 29.5 kDa proteins were expressed in insoluble forms in Escherichia coli to levels of 15% and 25% of the total cell proteins, respectively. Both the variant molecules reacted equally well with the antisera raised in rabbit against the native protein. PstS1 and tnPstS1 were evaluated through ELISA against plasma samples from 160 culture positive tuberculosis patients and 40 healthy controls. With tnPstS1 43% of the patient samples were detected positive for the antibody as compared to only 36% in the case of the native PstS1. Data for the secondary structures of the native and the truncated variants as obtained by circular dichroism agreed with the known 3-D structure of the native protein and the predicted structure of the truncated version, respectively. The results show that the truncated tnPstS1 is more efficient as compared to the native PstS1 for use as a serodiagnostic agent.

Production enhancement and refolding of caprine growth hormone expressed in Escherichia coli.

This study describes comparison between IPTG and lactose induction on expression of caprine growth hormone (cGH), enhancing cell densities of Escherichia coli cultures and refolding the recombinant cGH, produced as inclusion bodies, to biologically active state. 2-3 times higher cell densities were obtained in shake flask cultures when induction was done with lactose showing almost same level of expression as in case of IPTG induction. With lactose induction highest cell densities were achieved in TB (OD(600) 16.3) and M9NG (OD(600) 16.1) media, producing 885 and 892 mg cGH per liter of the culture, respectively. Lactose induction done at mid-exponential stage resulted in a higher cell density and thus higher product yield. cGH over-expressed as inclusion bodies was solubilized in 50 mM Tris-Cl buffer (pH 12.5) containing 2 M urea, followed by dilution and lowering the pH in a step-wise manner to obtain the final solution in 50mM Tris-Cl (pH 9.5). The cGH was purified by Q-Sepharose chromatography followed by gel filtration with a recovery yield of 39% on the basis of total cell proteins. The product thus obtained showed a single band by SDS-PAGE analysis. MALDI-TOF analysis showed a single peak with a mass of 21,851 dalton, which is very close to its calculated molecular weight. A bioassay based on proliferation of Nb2 rat lymphoma cells showed that the purified cGH was biologically active.

Improving sensitivity for serodiagnosis of tuberculosis using TB16.3-echA1 fusion protein

This study aimed at developing and assessing the fusion proteins with enhanced sensitivity to detect antibodies in plasma as a diagnostic method for tuberculosis. DNA fragments encoding TB16.3 and echA1 gene regions corresponding to proteins TB16.3 and echA1 from Mycobacterium tuberculosis were amplified through PCR. Through a series of restrictions and ligations two novel fusion constructs TB16.3-echA1 and TB16.3-tnPstS1 were produced and expressed in Escherichia coli. These were screened for detection of antibodies in human plasma. The individual antigens TB16.3, echA1 and tnPstS1 and the fusion protein TB16.3-tnPstS1 and TB16.3-echA1 showed sensitivities of 29%, 25.5%, 42.8%, 40.0% and 47.2%, respectively. Lower sensitivity in case of TB16.3-tnPstS1 seems to be due to the structural arrangement between the two proteins, which is likely to mask several of their epitopes. The higher sensitivity of TB16.3-echA1 appears to be due to lesser interaction between the two proteins thus allowing free availability of epitopes for binding antibodies. 64% of TB patients were found positive for either one of the two fusion proteins TB16.3-echA1 and TB16.3-tnPstS1. This study indicates that the novel fusion protein TB16.3-echA1 has a potential in serodiagnosis of TB with improved sensitivity and reliability.

Fusion of selected regions of mycobacterial antigens for enhancing sensitivity in serodiagnosis of tuberculosis

Serodiagnosis of tuberculosis requires detection of antibodies against multiple antigens of Mycobacterium tuberculosis, because antibody profiles differ among the patients. Using fusion proteins with epitopes from two or more antigens would facilitate in the detection of multiple antibodies. Fusion constructs tn1FbpC1-tnPstS1 and tn2FbpC1-tnPstS1 were produced by linking truncated regions of variable lengths from FbpC1 to the N-terminus of the truncated PstS1. Similarly a truncated fragment of HSP was linked to the N-terminus of a truncated fragment from FbpC1 to produce tnHSP-tn1FbpC1. ELISA analysis of the plasma samples of TB patients against tn2FbpC1-tnPstS1 showed 72.2% sensitivity which is nearly the same as the expected combined value for the two individual antigens. However, the sensitivity of tn1FbpC1-tnPstS1 was lowered to 60%. tnHSP-tn1FbpC1 showed 67.7% sensitivity which is slightly less than the expected combined value for the two individual antigens, but still significantly higher than that of each of the individual antigen. Data for secondary structure analysis by CD spectrometry was in reasonable agreement with the X-ray crystallographic data of the native proteins and the predicted structure of the fusion proteins. Comparative molecular modeling suggests that the epitopes of the constituent proteins are better exposed in tn2FbpC1-tnPstS1 as compared to those in tn1FbpC1-tnPstS1. Therefore, removal of the N-terminal non-epitopic region of FbpC1 from 34-96 amino acids seems to have unmasked at least some of the epitopes, resulting in greater sensitivity. The high level of sensitivity of tn2FbpC1-tnPstS1 and tnHSP-tn1FbpC1, not reported before, shows that these fusion proteins have great potential for use in serodiagnosis of tuberculosis.

Influence of transposition and insertion of additional binding domain on expression and characteristics of xylanase C of Clostridium thermocellum.

Clostridium thermocellum encodes a xylanase gene (xynC) which is the major component of its cellulosome. XynC is a multidomain enzyme comprising of a substrate binding domain at the N-terminal followed by the catalytic domain and a dockerin domain. To study the influence of binding domain on activity, stability and expression of the enzyme the protein with the binding domain at C-terminal (XynC-CB), and the one with the binding domain at both N- and C-terminal (XynC-BCB) were expressed in E. coli. Recombinant plasmids, pXynC-CB and pXynC-BCB were constructed by inserting the corresponding gene in pET22b(+). XynC-CB and XynC-BCB were expressed at levels around 30% and 33% of the total E. coli cell proteins, respectively, while losing 40% and 20% of their activities at 70°C for 120 min, respectively. The specific activities of XynC-CB, XynC-BCB were 76 and 98 U mg(-1), while the activities on equimolar basis were 4410 and 7450 U μM(-1) against birchwood xylan, respectively. Their overall activities produced in the culture were 3660 and 5430 U L(-1) OD(600)(-1). Substrate binding studies showed that in case of XynC-C 51% of the activity remained unbound to birchwood xylan, whereas in the cases of XynC-BC, XynC-CB and XynC-BCB the activities left unbound were 33%, 32% and 12%, respectively, under the assay conditions used. Similar binding values were obtained in the case of oat spelt xylan. K(m) values for XynC-CB and XynC-BCB against birchwood xylan were found to be 3.1 and 1.47 mg ml(-1), respectively. Thus addition of a second carbohydrate binding domain at the C-terminal of the catalytic domain enhances activity, substrate affinity as well as thermostability.