Secreted protein markers in oral squamous cell carcinoma (OSCC).

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a main cause of oral cancer mortality and morbidity in central south Asia. To improve the clinical outcome of OSCC patients, detection markers are needed, which are preferably non-invasive and thus independent of a tissue biopsy. METHODS: In the present study, we aimed to identify robust candidate protein biomarkers for non-invasive OSCC diagnosis. To this end, we measured the global protein profiles of OSCC tissue lysates to matched normal adjacent mucosa samples (n = 14) and the secretomes of nine HNSCC cell lines using LC-MS/MS-based proteomics. RESULTS: A total of 5123 tissue proteins were identified, of which 205 were robustly up- regulated (p-value < 0.01, fold change > + 2) in OSCC-tissues compared to normal adjacent tissues. The biological process "Secretion" was highly enriched in this set of proteins. Other upregulated biological pathways included "Unfolded Protein Response", "Spliceosomal complex assembly", "Protein localization to endosome" and "Interferon Gamma Response". Transcription factor analysis implicated Creb3L1, ESRRA, YY, ELF2, STAT1 and XBP as potential regulators. Of the 205 upregulated tissue proteins, 132 were identified in the cancer cell line secretomes, underscoring their potential use as non-invasive biofluid markers. To further prioritize our candidate markers for non-invasive OSCC detection, we integrated our data with public biofluid datasets including OSCC saliva, yielding 25 candidate markers for further study. CONCLUSIONS: We identified several key proteins and processes that are associated with OSCC tissues, underscoring the importance of altered secretion. Cancer-associated OSCC secretome proteins present in saliva have potential to be used as novel non-invasive biomarkers for the diagnosis of OSCC.

SUMO-fusion and autoinduction-based combinatorial approach for enhanced production of bioactive human interleukin-24 in Escherichia coli.

High-level production of recombinant human interleukin-24 (IL-24), a multifunctional immunomodulatory cytokine, has been challenging due primarily to its aggregation as inclusion bodies in the bacterial host while persistent poor-expression in the insect/mammalian expression systems. The present study presents a robust, vector-host combination (pE-SUMO-IL24), auto-inducible medium (YNG/M9NG), and a simple purification scheme for soluble, bioactive, and cost-effective production of native-like IL-24 (nIL-24) in Escherichia coli. The final protein yield, following a three-step purification scheme (IMAC, SEC, dialysis), was 98 mg/L in shake-flask culture (with scale-up potential), which was several folds higher than reported earlier. In vitro cytotoxicity assays with HeLa and HCT116 cancer cell lines (performed using different concentrations of nIL-24) and the fluorescence activated cell sorting analysis (FACS) revealed a dose- and concentration-dependent increase in the population of pro-apoptotic cells with concomitant, statistically significant drop in the number of cells existent at Go/G1-, S-, and G2/M-phases (P < 0.002). The bioactive nIL-24, developed through this study, holds promise for use in further functional characterizations/applications. KEY POINTS: • Yeast SUMO fusion partner at N-terminus for improved solubility of an otherwise insoluble IL-24 in E. coli. • Enhanced cell densities with concomitant several-fold increase in protein yield by lactose-inducible media. • Improved inhibition of cervical and colorectal carcinomas by native-like nIL-24 compared with Met-containing IL. • Heterologous nIL-24 may enable better understanding of the functional intricacies linked up with its unique cancer-specific features. Graphical abstract.

Potential of multi-component antigens for tuberculosis diagnosis.

Tuberculosis is one of the top ten causes of deaths worldwide. The cause of tuberculosis is a bacterium, Mycobacterium tuberculosis, which has been surviving for centuries. Immunological tests based on detecting the presence of antibodies in the sera of active TB patients against various antigens of M. tuberculosis are useful for diagnosis of TB and offer simple, rapid and cost effective methods most suitable for poor and developing countries. Several recombinant antigens have been reported so far with varying sensitivity individually, yet none had shown sensitivity higher enough to be used in a commercial test. There is a trend of utilizing recombinant DNA technology to make polypeptide chain with two or more different antigenic regions, in order to increase the diagnostic efficiency. In this review, we have made an attempt to combine current studies on the usefulness of the multi-component Mycobacterium tuberculosis antigens in serological tests for the diagnosis of tuberculosis.

Deep sequencing of Escherichia coli exposes colonisation diversity and impact of antibiotics in Punjab, Pakistan.

Multi-drug resistant (MDR) E. coli constitute a major public health burden globally, reaching the highest prevalence in the global south yet frequently flowing with travellers to other regions. However, our comprehension of the entire genetic diversity of E. coli colonising local populations remains limited. We quantified this diversity, its associated antimicrobial resistance (AMR), and assessed the impact of antibiotic use by recruiting 494 outpatients and 423 community dwellers in the Punjab province, Pakistan. Rectal swab and stool samples were cultured on CLED agar and DNA extracted from plate sweeps was sequenced en masse to capture both the genetic and AMR diversity of E. coli. We assembled 5,247 E. coli genomes from 1,411 samples, displaying marked genetic diversity in gut colonisation. Compared with high income countries, the Punjabi population generally showed a markedly different distribution of genetic lineages and AMR determinants, while use of antibiotics elevated the prevalence of well-known globally circulating MDR clinical strains. These findings implicate that longitudinal multi-regional genomics-based surveillance of both colonisation and infections is a prerequisite for developing mechanistic understanding of the interplay between ecology and evolution in the maintenance and dissemination of (MDR) E. coli.

Circulating miR-146a expression as a non-invasive predictive biomarker for acute lymphoblastic leukemia.

Dysregulation of non-coding microRNAs during the course of tumor development, invasion and/or progression to the distant organs, makes them a promising candidate marker for the diagnosis of cancer and associated malignancies. This exploratory study aims at evaluating the usefulness of plasma concentration of circulating mir-146a as a non-invasive biomarker for acute lymphoblastic leukemia (ALL). Total RNA including miRNA was isolated from 110 plasma samples of patients (n = 66), healthy controls (n = 24) and follow up (n = 20) cases and reverse transcribed. Relative concentrations were assessed using real-time quantitative PCR and fold-change was calculated by 2-ΔΔCt method. Finally, relative concentrations were correlated to clinicopathological factors. Patients (n = 66) were analyzed to determine fold expression of miR-146a in plasma samples of ALL. Before chemotherapy, pediatric (n = 42) and adult (n = 24) showed overexpression of miR-146a compared with healthy controls (P < 0.0001). There was no effect of age and gender on mir-146a expression in plasma. mirR-146a expression was independent of clinical and hematological features. Moreover, miR-146a levels in plasma of paired samples (n = 20) after treatment showed significant decrease in expression (P < 0.001). Expression of plasma miR-146a may be utilized as non-invasive marker to diagnose and predict prognosis in pediatric and adult patients with ALL. Moreover predicted targets may be utilized for ALL therapy in future.

Balance between Protection and Pathogenic Response to Aerosol Challenge with Mycobacterium tuberculosis (Mtb) in Mice Vaccinated with TriFu64, a Fusion Consisting of Three Mtb Antigens.

Tuberculosis vaccines capable of reducing disease worldwide have proven difficult to develop. BCG is effective in limiting childhood disease, but adult TB is still a major public health issue. Development of new vaccines requires identification of antigens that are both spatially and temporally available throughout infection, and immune responses to which reduce bacterial burden without increasing pathologic outcomes. Subunit vaccines containing antigen require adjuvants to drive appropriate long-lived responses. We generated a triple-antigen fusion containing the virulence-associated EsxN (Rv1793), the PPE42 (Rv2608), and the latency associated Rv2628 to investigate the balance between bacterial reduction and weight loss in an animal model of aerosol infection. We found that in both a low pattern recognition receptor (PRR) engaging adjuvant and a high PRR-engaging adjuvant (MPL/TDM/DDA) the triple-antigen fusion could reduce the bacterial burden, but also induced weight loss in the mice upon aerosol infection. The weight loss was associated with an imbalance between TNFα and IL-17 transcription in the lung upon challenge. These data indicate the need to assess both protective and pathogenic responses when investigating subunit vaccine activity.

Improvement in activity of cellulase Cel12A of Thermotoga neapolitana by error prone PCR.

Using multi-step error prone PCR (ep-PCR) of the gene encoding endoglucanase Cel12A (27 kDa) from Thermotoga neapolitana, mutants were obtained with many fold increase in the enzyme activity. The best mutant (C6, N47S/E57 K/ V88A/S157 P/K165 H) obtained after four rounds of ep-PCR showed 2.7-, 5- and 4.8-fold increase in activity against CMC, RAC and Avicel, respectively, compared with the wild type enzyme. The other characteristics of the mutated enzyme with respect to stability, optimum working pH and temperature were comparable to the wild type enzyme.C6 mutant showed higher binding efficiency towards the rice straw (∼50%) than the wild type (∼41%). The structural information obtained from the protein docking of the wild type Cel12A and its mutant showed that E57 K improved the binding affinity between enzyme and ligand by producing conformational changes in the catalytic cavity. The other mutations can facilitate the enzyme-substrate binding interactions to enhance catalytic activity although they are not directly involved in catalysis. The wild type and mutant enzyme produce cellobiose as the major products for both soluble and insoluble substrates, suggesting that this enzyme should be a cellobiohydrolase instead of endoglucanase as previously reported.

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.

Health improvement of human hair and their reshaping using recombinant keratin K31.

Hair, being one of the most important components of the beauty care processes, attracts the use of a variety of hair treating cosmetics. Conventional hair treating cosmetics are not satisfactory for one reason or the other. Commercially used keratins are isolated from wool or chicken feathers. As these lack complete sequence identity with human hair keratin, are likely to be less efficient than the human hair keratin. K31, a type I acidic keratin, is a major protein of human hair keratin complex and it is essential for maintaining the hair tensile strength. In this study keratin K31 (46 kDa) gene was expressed in Escherichia coli at a level of approximately 35% of the total cell proteins. The protein, however, was expressed as insoluble inclusion bodies. The expressed protein was refolded and purified by FPLC using an anion-exchange column. The CD analysis results showed that the K31 was properly refolded. MALDI-TOF mass spectroscopy showed the characteristics expected for human K31 keratin. The refolded and partially purified K31 protein, when applied on chemically damaged hairs, increased the diameter of the hair up to 49%. The mechanical strength of the bleached hair increased by almost 2 fold after a single treatment of K31. The protein also straightened curly hair efficiently on a single treatment for one hour. Application of K31 resulted in marked improvements in smoothness, diameter and mechanical strength of the damaged hair.

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.

Removal and recovery of uranium from aqueous solutions by Trichoderma harzianum.

Removal and recovery of uranium from dilute aqueous solutions by indigenously isolated viable and non-viable fungus (Trichoderma harzianum) and algae (RD256, RD257) was studied by performing biosorption-desorption tests. Fungal strain was found comparatively better candidate for uranium biosorption than algae. The process was highly pH dependent. At optimized experimental parameters, the maximum uranium biosorption capacity of T. harzianum was 612 mg U g(-1) whereas maximum values of uranium biosorption capacity exhibited by algal strains (RD256 and RD257) were 354 and 408 mg U g(-1) and much higher in comparison with commercially available resins (Dowex-SBR-P and IRA-400). Uranium biosorption by algae followed Langmuir model while fungus exhibited a more complex multilayer phenomenon of biosorption and followed pseudo-second-order kinetics. Mass balance studies revealed that uranium recovery was 99.9%, for T. harzianum, and 97.1 and 95.3% for RD256 and RD257, respectively, by 0.1M Hydrochloric acid which regenerated the uranium-free cell biomass facilitating the sorption-desorption cycles for better economic feasibility.

Identification of actin beta-like 2 (ACTBL2) as novel, upregulated protein in colorectal cancer.

Early diagnosis of colorectal cancer (CRC) can be of value for increasing the survival rate of patients. Recently, proteomic strategies to identify markers for the diagnosis of cancer at an early stage have been employed with noteworthy results. To extend these studies, we utilized two dimensional gel electrophoresis and mass spectrometry for expression profiling of proteins extracted from the freshly frozen human colorectal cancer tissue specimens and the comparable regions of adjacent normal mucosa (serving as controls). Four gel spots were determined to be differentially stained between the tumor and the control samples on a consistent basis. Following mass spectrometric analysis of these spots, six proteins were identified; five of these had previously been reported to be associated with colorectal cancer. One protein actin beta-like 2 (ACTBL2), not linked with colorectal cancer in the earlier reports, was however found to be at higher abundance in colorectal tumor samples both by proteomics and immunohistochemistry analysis. Thus ACTBL2 association and differential upregulation in colorectal cancer is novel, and as such may contribute to our understanding of the colorectal carcinogenesis and potentially serve a function in developing markers for colorectal cancer. BIOLOGICAL SIGNIFICANCE: Colorectal cancer (CRC) is a major cause of death world-wide and good markers for early detection are lacking. In this study we conducted a proteomic analysis of tumor vs. normal tissue. We corroborated the finding of a number of previously identified proteins associated with CRC and more importantly identified a novel protein, ACTBL2, which we demonstrated to be upregulated in CRC. As additional proteins associated with CRC are identified the potential for developing panels of markers may be realized with better outcomes in early cancer detection.

Removal and recovery of lead(II) from single and multimetal (Cd, Cu, Ni, Zn) solutions by crop milling waste (black gram husk).

The study reports removal of heavy metals when present singly or in binary and ternary systems by the milling agrowaste of Cicer arientinum (chickpea var. black gram) as the biosorbent. The biosorbent removed heavy metal ions efficiently from aqueous solutions with the selectivity order of Pb>Cd>Zn>Cu>Ni. The biosorption of metal ions by black gram husk (BGH) increased as the initial metal concentration increased. Biosorption equilibrium was established within 30 min, which was well described by the Langmuir and Freundlich adsorption isotherms. The maximum amount of heavy metals (qmax) adsorbed at equilibrium was 49.97, 39.99, 33.81, 25.73 and 19.56 mg/g BGH biomass for Pb, Cd, Zn, Cu and Ni, respectively. The biosorption capacities were found to be pH dependent and the maximum adsorption occurred at the solution pH 5. Efficiency of the biosorbent to remove Pb from binary and ternary solutions with Cd, Cu, Ni and Zn was the same level as it was when present singly. The presence of Pb in the binary and ternary solutions also did not significantly affect the sorption of other metals. Breakthrough curves for continuous removal of Pb from single, binary and ternary metal solutions are reported for inlet-effluent equilibrium. Complete desorption of Pb and other metals in single and multimetal solutions was achieved with 0.1 M HCl in both shake flask and fixed bed column studies. This is the first report of removal of the highly toxic Pb, Cd, and other heavy metals in binary and ternary systems based on the biosorption by an agrowaste. The potential of application for the treatment of solutions containing these heavy metals in multimetal solutions is indicated.

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.

Free and total prostate specific antigen in benign prostate hyperplasia and prostate cancer.

OBJECTIVE: To record the levels of PSA in the sera of prostate cancer (CaP) and benign prostatic hyperplasia (BPH) cases. Free PSA/total PSA as percentage was also calculated in order to evaluate its utility in differentially diagnosing BPH and CaP. DESIGN: A cross-sectional, case control study. PLACE AND DURATION OF STUDY: Shaikh Zayed Hospital and Mayo Hospital, Lahore from August 2002 to March 2003. MATERIALS AND METHODS: A group of 108 male subjects, including one-third of each of biopsy-confirmed prostate cancer cases, BPH cases and asymptomatic controls of matching age were studied. PSA and Free PSA were determined by ELISA using commercially available assay kits. RESULTS: Mean PSA was found to be highest in CaP cases (41.9 +/- 38.7 ng/ml), lower in the BPH cases (13.5 +/- 10.5 ng/ml), while it was lowest in the control subjects (5.7 +/- 4.4 ng/ml). Moreover, it was observed that a majority of the CaP cases had serum PSA >20 ng/ml, 50% of BPH cases had serum PSA in the 'gray zone' (4.1-20 ng/ml), while majority of controls had serum PSA in the 'normal' range (0 - 4 ng/ml). Using a free- PSA "cut-off" of 18% to differentiate between benign and malignant prostate enlargement, it was found that 80% of the CaP cases had F/T% <18, while 75% of the BPH cases had F/T%>18. The percent free-PSA test to differentially diagnose BPH and CaP in the 'gray zone' was found to have a sensitivity of 86% and a specificity of 94%. CONCLUSION: Using a cutoff of 18%, the free-PSA test significantly improved the differential diagnosis of BPH and CaP in the 'gray zone' as compared to the use of total PSA alone in the study group.

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.

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.

Proteomic identification of human urinary biomarkers in diabetes mellitus type 2.

BACKGROUND: During the proteomic era, one of the most rapidly growing areas in biomedical research is biomarker discovery, particularly using proteomic technologies. The urinary proteome is known to be a valuable field of study and has become one of the most attractive subdisciplines in clinical proteomics for human diseases. We have described the levels of protein biomarkers specific to diabetes mellitus type 2 in the Pakistani population using proteomic technology. METHODS: One hundred type 2 diabetes patients with 50 age- and sex-matched normal healthy controls were recruited from Sheikh Zayed Hospital, Lahore, Pakistan. Urinary proteins were analyzed by two-dimensional liquid chromatography, using chromatofocusing in the first dimension and reverse-phase chromatography in the second, followed by mass spectrometric analysis. Levels of the proteins, which were found to vary in the diabetes type 2 patients compared to the controls, were then determined by enzyme-linked immunosorbent assay in all the samples. RESULTS: Levels of transthyretin, α-1-microglobulin/bikunin precursor, and haptoglobin precursor decreased by 30.8%, 55.2%, and 81.45%, whereas levels of albumin, zinc α2 glycoprotein, retinol binding protein 4, and E-cadherin increased by 486.5%, 29.23%, 100%, and 693%, respectively, in the diabetes patients compared to the controls. CONCLUSIONS: Variation in the levels of these identified protein biomarkers have been reported in other pathological states. Assessment of the levels of these biomarkers will be helpful not only in early diagnosis but also in prognosis of diabetes mellitus type 2.

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.

Enhanced expression and activity yields of Clostridium thermocellum xylanases without non-catalytic domains.

Two major xylanase components, XynC and XynZ from the anaerobic thermophilic bacterium, Clostridium thermocellum cellulosome were cloned and expressed with and without non-catalytic domains in E. coli. Two constructs of XynC, one with its cellulose binding domain and the catalytic domain (pXynC-BC) and the other with only the catalytic domain (pXynC-C) were produced. For XynZ the constructs produced were pXynZ-BDC, which included the dockerin domain, and pXynZ-C, which did not. E. coli cells transformed with pXynC-BC or pXynZ-BDC gave xylanase expression of 30% and 25% total cell proteins, respectively. Transformation of E. coli cells with the constructs carrying only the catalytic domains gave expression levels of approximately 45% in each case. The specific activities of XynC with and without the non-catalytic domains were similar, but for XynZ the specific activity of the enzyme without the non-catalytic domains was approximately 5-fold greater than that of the intact enzyme. The total activity increased from 1925Ul(-1)OD(600)(-1) for XynC-BC to 3050Ul(-1)OD(600)(-1) for XynC-C. However, the overall increase in activity was approximately 9-fold higher for XynZ-C (32,900Ul(-1)OD(600)(-1)) versus XynZ-BDC (3665Ul(-1)OD(600)(-1)). Both the enzymes with and without non-catalytic domains were found to be quite stable over a broad pH range (pH 4-9). XynZ-C was more thermostable than XynZ-BDC as it retained 87% of xylanase activity when incubated at 70 degrees C for 2h as compared to 42% for XynZ-BDC. However, XynC-BC retained 70% activity on incubation at 70 degrees C for 2h but XynC-C lost all activity under the same conditions. K(m) values for XynC-BC and XynC-C determined on soluble xylan were 3.1 and 3.6 mg ml(-1), respectively, whereas these values for XynZ-BDC and XynZ-C were 33.3 and 15.4 mg ml(-1), respectively. Thus the production of xylanase activity by expressing only the catalytic domains of XynC and XynZ is significantly enhanced.