Rates of growth (OD678/day

Growth

Experimental Set up

BIOMASS PRODUCTION: ROLE OF N-P CONCENTRATION AND RATIOS

Soil pH and electrical conductivity (EC)

Experiment 2: Assessing the impacts of elevated temperature and N levels on yield and nutrient uptake in rice

Experiment 1: Assessing the impacts of elevated CO2and N levels on yield and nutrient uptake in rice

Experimental Design and Treatments

Temperature Gradient Tunnels (TGT)

Yield and its component traits:

Differences among cultivars for yield, biomass and HI

Kernel hardness

SDS-sedimentation test

Estimation of total N% of wheat grainsand straw

End use Quality parameters

Chlorophyllcontent

Root length (cm) and Root weight (mg)

Coleoptile length(cm)

Stomata / cm2

Leaf area index (LAI)

Physiological parameters

Laboratory observations

Days to physiological maturity

Static and shaken condition

Screening for proteolytic activity

Vaccination improved the Chemokine receptor CCR1, CCR3, CCR9 and Toll like receptor TLR2, TLR4 and TLR9 expression in HBsAgpositive newborns compared to healthy newborns.

IFN γ production by CD8 T cells upon stimulation with PMA and viral peptides

Pre-vaccination:Lower Chemokine and Toll like receptor expression in HBsAgpositive newborns:

T cell phenotypic distribution in HBsAgPositive, HBsAgNegative from HBsAg positive mothers and healthy newborns.

Sample Collection

1X TBS

Immunofluorescence

Secondary antibodies

Animals

GFP-β-catenin T41A mutant

Transfection of short interfering RNA (siRNA) and plasmid

Mammalian cell lines

Assay of IFN-yand IL-10 using sandwich ELISA

Anti-Stm and anti-E coli /gG subclass assay

Mice

Cellline

Extraction of Tannin

Extraction

Procedure

Standard curve of sugar

Extraction and determination of protein

Standard curve of protein

Reagents

Estimation of protein

Standard curve of ascorbic acid

Fresh and Dry weight

Dilution Formula

Cd (NO3)2

ZnSO4

Preparation of ppm solution

Youman's modified medium

Histopathological studies

Bacterial Cultures

Preparation of ultra competent E. coli

Antibiotics

Monoclonal Antibodies against the Human Glycophorin-

Monoclonal Antibodies against the Human Glycophorin

Isolation of endothelial cells from rat aorta

Procedur

Procedur

Procedure

Procedure

Reagent

Total antioxidant activit

Procedur

Catalyst Preparation

Catalyst Preparation

Catalyst Preparation

Single Cell Test

Electrochemical Measuremen

Physical Characterization

Catalyst Preparation

Material

ELECTROGENERATION OF SEAWATER ON ALCOHOL OXIDATION IN AN NON-MEMBRANE POWER SYSTEM

Total dissolved solids

High Performance Liquid Chromatography (HPLC) analysis

High Performance Thin Layer Chromatography (HPTLC) analysis

. Thin Layer Chromatography (TLC) analysis

Preliminary phytochemical screening (Ali, 1998; Evans, 2002)

Microbial Contamina

Foaming Index

pH values

Ash values

Extractive value

. Physico-chemical standardization

Standardization of ethanolic extract of E.ribes (WHO, 1998; IndianPharmacopoeia, 1996)

Estimation of Stevioside

Estimation of Steviol

Estimation of total phenols

Somatotype Categories:

somatotype Categories:

somatotype Categories:

Range of Component Ratings:

Somatocharts:

Mean Somatotypes:

SOMATOTYPE DISTRIBUTION OF RURAL SAMPLE

Somatocharts:

Balanced mesomorph:

Overnight grown primary culture of E. coli cells (1 % v/v final concentration) was inoculated into 1 litre of LB media containing antibiotics. Culture was incubated at 37 oc at 200 rpm. Growth was monitored by measuring absorbance of E. coli broth at 600 nm. Culture was induced by adding 1 mM IPTG at an OD of 0.6 and was harvested after 4 hrs of induction. Samples were taken on an hourly basis after induction to check the kinetics of protein expression. Un-induced and induced E. coli cells were analyzed by SDS-PAGE to check the expression of recombinant protein.

Growth and expression of recombinant proteins in E. coli cells

The [3-PMB and a-PMB chains were eluted with a linear gradient of 500 ml each of 0.01 M potassium phosphate buffer (pH 6.5) and 0.015 M potassium phosphate buffer (pH 8.5) at a flow rate of 50 ml/hour. The chains were separately concentrated using Centriprep concentrators (Amicon) and stored in liquid nitrogen till further use

The heme bound a and ~ subunits were obtained as described by Bucci (1981 ). Briefly, hemoglobin was reacted with PMB in an eight fold molar excess (8 moles of PMB per mole of hemoglobin). The reaction mixture was dialyzed extensively against 0.01 M potassium phosphate buffer (pH 6.5) and then loaded onto a CM52 column (30cm x !Scm) that was pre-equilibrated with the same buffer.

Separation of the a and f3 subunits of hemoglobin

Paduans

Riding over the hills, and eating their fill,7 lying a little too long; these things are, perhaps, enough to explain what happened. How­ever, that may be: they woke suddenly from a sleep they had never meant to take. The standing stone was cold, and it cast a long pale shadow. The sun was gleaming through the mist; north, south, and east, the fog was thick, cold and white. The air was silent, heavy and chill.The hobbits8 sprang to their feet in alarm, and ran to the western rim. They found that they were upon an island in the fog. Even as they looked out in dismay towards the setting sun, it sank before their eyes into a white sea, and a cold grey shadow sprang up in the East behind. The fog rolled up to the walls and rose above them, and as it mounted it bent over their heads until it became a roof. They felt as if a trap was closing about them. They packed up as quickly as their chilled fingers would work.Soon they were leading their ponies in single file9 over the rim and down the long northward slope of the hill, down into a foggy sea. As they went down the mist became colder and damper, and their hair hung lank and dripping on their foreheads. When they reached the bottom it was so cold that they halted and got out cloaks and hoods, which soon became bedewed with grey drops. Then, mounting their ponies, they went slowly on again. To prevent their getting separated and wandering in different directions they went in file, with Frodo leading. Suddenly Frodo saw a hopeful sign. On either side ahead a darkness began to loom through the mist; and he guessed that they were at last approaching the gap in the hills. 'Come on! Follow me!' he called back over his shoulder, and he hurried forward. His pony reared, and he fell off. When he looked back he found that he was alone: the others had not fol­lowed him.

Riding over the hills, and eating their fill,7 lying a little too long; these things are, perhaps, enough to explain what happened. How­ever, that may be: they woke suddenly from a sleep they had never meant to take. The standing stone was cold, and it cast a long pale shadow. The sun was gleaming through the mist; north, south, and east, the fog was thick, cold and white. The air was silent, heavy and chill.The hobbits8 sprang to their feet in alarm, and ran to the western rim. They found that they were upon an island in the fog. Even as they looked out in dismay towards the setting sun, it sank before their eyes into a white sea, and a cold grey shadow sprang up in the East behind. The fog rolled up to the walls and rose above them, and as it mounted it bent over their heads until it became a roof. They felt as if a trap was closing about them. They packed up as quickly as their chilled fingers would work.Soon they were leading their ponies in single file9 over the rim and down the long northward slope of the hill, down into a foggy sea. As they went down the mist became colder and damper, and their hair hung lank and dripping on their foreheads. When they reached the bottom it was so cold that they halted and got out cloaks and hoods, which soon became bedewed with grey drops. Then, mounting their ponies, they went slowly on again. To prevent their getting separated and wandering in different directions they went in file, with Frodo leading. Suddenly Frodo saw a hopeful sign. On either side ahead a darkness began to loom through the mist; and he guessed that they were at last approaching the gap in the hills. 'Come on! Follow me!' he called back over his shoulder, and he hurried forward. His pony reared, and he fell off. When he looked back he found that he was alone: the others had not fol­lowed him.

The clear cell-free supernatants were used as the source of crude recombinant xylanase.

Quantitative screening for determination of xylanase in shake flask

2 mL of an overnight culture of E. coli cells was inoculated into 100 mL LB medium and incubated with vigorous shaking at 30 °C until A600 of 0.8 was reached. •Cells were collected in 50 mL plastic (Falcon) tubes, cooled for 15 min on ice and centrifuged in a pre-cooled centrifuge (4,000 rpm for 10 min at 4 °C). •The pellet was suspended in 20 mL of ice-cold 50 mM CaCl2-15% glycerol solution, maintained on ice for 15 min and centrifuged again at 4,000 rpm for 10 min at 4 °C. •Pellet was resuspended in 2 mL of ice-cold 50 mM CaCl2-15 % glycerol solution, kept on ice for 30 min and aliquoted in 400 μL in microcentrifuge tubes. These were stored at -80 °C until required.

Preparation of calcium-competent cells

Two hundred μL of alkaline-SDS solution was added to the above suspension, mixed by inverting the tubes up and down 3 times and incubated for 5 min at room temperature. ƒTo the above mixture, 250 μL of 3 M Na-acetate (pH 4.8) was added, mixed by inverting the tubes up and down 3 times, and centrifuged at 12,000 x g for 10 min. ƒThe supernatant was collected in another micro centrifuge tube (MCT), 200 μL of phenol:chloroform solution was added, inverted two times and centrifuged at 12, 000 x g for 8 min at room temperature. ƒThe aqueous phase was transferred to new tubes and 500 μL of chilled (-20 °C) ethanol (96 %) was added. ƒThe tubes were centrifuged at 13,000 x g for 25 min at 4 °C, supernatant discarded and pellet dried for 15 min at room temperature. ƒThe pellet was washed with 500 μL of chilled 70 % (v/v) ethanol and centrifuged at 13, 000 rpm for 4 min at 4 °C. ƒThe pellet was dried at room temperature and dissolved in 50 μL of 1X TE buffer (pH 8.0) containing RNase and stored at -20 °C till further use.

The cells of E. coli DH10B having p18GFP vector were cultivated for overnight at 37 °C in LB medium containing ampicillin (100 μg mL-1). ƒThe E. coli culture having p18 GFP vector (~1.5 mL) was taken in Eppendorf tubes and centrifuged at 10, 000 x g for 5 min. ƒThe pellet was homogenized by vortex mixing in 100 μL of homogenizing solution

Plasmid isolation from miniprep method

An attempt was made to study the effect of storage of DNA extracts on DNA yield and purity. The DNA extracts were centrifuged and the supernatants were dispensed into 2.0 mL Eppendorf tubes and stored at -20 oC for a month. DNA precipitation and its quantification were carried out at a week intervals.

Effect of storage on soil/sediment DNA extracts

Attempts have been made to amplify the signature sequences of bacterial, archaeal and fungal specific regions by using respective sets of primers shown in Table2.2. The reactions were carried out in 50 μL reaction mixtures in a Thermal Cycler (Bio-Rad, USA) using respective primers (Table 2.2). The PCR conditions were optimized as follows: for Bacterial 16S rDNA, initial denaturation of 3 min at 94 oC followed by 30 cycles of 30 sec at 93 oC, 60 sec at 55 oC and 90 sec at 72 oC; Archaeal 16S rDNA, 5 min at 95 oC, 35 cycles of 50 sec at 94 oC, 60 sec at 62 oC and 60 sec at 72 oC; fungal specific ITS regions, 3 min at 95 °C, 30 cycles of 60 sec at 94 °C, 56 °C at 45 sec and 50 sec at 72 °C. Final extension time was 7 min at 72 °C in all PCR runs. Amplifications were visualized on 1.2 % w/v agarose gels

PCR amplification of microbial population

Purity of the DNA extracted from various environmental samples was confirmed by subjecting the extracted DNA to restriction digestion. DNA was digested with Sau3AI (New England Biolabs). One μg of metagenomic DNA in 20 μL reaction mixture was treated with 0.5 U of Sau3AI and incubated at 37 °Cfor 10 min. The reaction was terminated at 80 °C for 20 min and the digested DNA was fractionated on 1.2 % (w/v) agarose gel.

Restriction digestion

VALIDATION OF METAGENOME OBTAINED BY THE PROTOCOL DEVELOPED IN THIS INVESTIGATION

as well as commercial methods (MN kit, Germany; Mo-Bio kit, CA, USA; Zymo soil DNA kit, CA, USA) according to the manufacturer’s protocols and compared in terms of DNA yield and purity.

The soil DNA from Pantnagar and Lonar soil samples were also extracted by various manual (Desai and Madamwar, 2007; Agarwal et al., 2001; Yamamoto et al., 1998

Comparison of yield and purity of crude DNA

Various strains of Escherchia coli (DH5α, XL1Blue, DH10B) were used as hosts for the propagation of recombinant vectors. In addition, Bacillus subtilis was used as a host for the expression of xylanase gene from the recombinant vector pWHMxyl. Different vectors used in this investigation are listed in

BACTERIAL STRAINS

Parasites from synchronized cultures were harvested at different time points of growth to obtain ring, trophozoite and schizont stage parasites. RNA was isolated from these stages by using RNAeasy kit (Qiagen) following manufacturer's protocol. The concentration of total RNA was determined by measuring the absorbance at 260 nm. Purity of nucleic acid preparations were determined by calculating OD26onm / OD28onm ratio, a value of near ~ 1.6-1.8 was taken as a standard of purity. To get stage specific cDNA from RNA, reverse transcription was performed using RT-PCR kit (Invitrogen) that contained random hexamers. Subsequently, the gene of interest was amplified using gene specific primers

Isolation of the parasite RNA

Human 0+ or AB+ RBC was obtained from a donor and mixed with heparin (50 units/ml of blood) and centrifuged at 500 g for 10 min with minimu1p. de-acceleration. The supernatant was removed carefully and the pelleted RBCS were washed 3 times with RPMI 1640 to remove serum and buffy coat. Equal amount of RPMI 1640 media was added to packed RBC volume to achieve 50% hem~tocrit and stored at 4°C till: further use

Preparation of RBCsfor culture

proteins of interest were pooled and 1 mM TCEP was added. The protein of interest was collected and stored at -80°C for further use after adding 1 mM TCEP.

The mutant proteins were expressed and purified analogous to wild type RaPt protein. Mutant clones pAC36, pAC50 and pAC38 were transformed in BL21 strain of E. coli. Analogous to the wild type RGPL protein the cells harbouring the mutant expression plasmids were cultured at 37°C to an O.D6oonm of 0.6 and uninduced at 30°C for 6-8 hrs. After harvesting, the cells were resuspended in lysis buffer (1 00 mM phosphate pH: 7 .0, I 0% glycerol) and disrupted using french press at 1100 psi pressure. Cell debris was removed by centrifugation at 50,000 g for 40 min at 4°C. 0. 75 ml L.1 of Ni2+ -NT A slurry was added to the supernatant and incubated at 4°C for 1 hr. This suspension was loaded onto a column working under gravity flow. The resin was washed with wash buffer (100 mM phosphate pH: 7.0, 10% glycerol and 5 mM imidazole) till all unbound proteins were removed. The protein was eluted using elution buffers containing increasing concentration of imidazole. Fractions containing the

Expression and purification of RcPL mutant proteins

Hair from the skin overlying the left and right dorsal flanks were removed using electrically operated razor. The skin overlying the abdomen was sterilized by wiping with 70% ethanol. Ketamine (1 00 mg/kg) and xylocaine (2%) (20 mg/kg) were mixed and administered intraperitoneally. The mice were returned to the cage and the onset of anesthetic effect was monitored. The mice were considered to be in surgical anesthesia when there was loss of palpebral reflex, righting reflex, and toe pinch reflex. Respiratory rate and heart rate were monitored continuously.

General anesthesia:

treatment were harvested by centrifugation at 250 x g for 5 min following which they were resuspended in 1x PBS (pH 7.5). PI was added at a final concentration of 1 J.tg/mL and incubated for 5 minutes following which the cells were pelleted by centrifugation and washed once with PBS. These cells were analyzed for uptake of PI by either flow cytometry in FL2 channel (570 nm) or by fluorescence microscopy using a G2A filter block.

Propidium iodide (PI) is a DNA intercalating fluorescent dye which is excluded by viable cells with intact membranes, however, dead and dying cells with damaged membranes take up the dye. To assess viability, cells after appropriate

population was determined by analyzing cells immunostained with an antibody against CD14 conjugated to FITC and the purity obtained was approximately 85% monocytes, the remaining being lymphocytes. The monocytes were further cultured in the presence of human AB serum for 7 days to allow differentiation to macrophages. At the end of 7 days post-isolation, greater than 95% of cells in culture are monocytes, with the majority of lymphocytes undergoing neglect induced death.

Peripheral blood (30 mL) was collected by venipuncture from healthy male volunteers after obtaining an informed consent and in accordance to the regulations of the Institutional Human Ethics Committee (National Institute of Immunology, New Delhi, India). The peripheral blood mononuclear cell (PBMC) population was isolated by density gradient centrifugation using Histopaque 1077, where, human whole blood was layered on Histopaque 1077 and centrifuged at 400 x g for 35 min at 25°C. The mononuclear cell population was isolated from the plasma-histopaque interface, and the monocytes were further purified by washing off the non-adherent cells after incubating the total PBMC for 1 h at 3 7°C. The homogeneity

Peripheral blood monocy.te isolation and macrophage differentiation

Textpresso

PubTator

include SORTING, that sorts, packs and assesses the quality of the experimentally measured diffraction data, and is run in the first step. The program TABLING calculates the continuous Fourier coefficients from the model placed in the artificial cell. The cross-rotation function is carried out by the program ROTING, which uses Crowther's algorithm (Crowther, 1972). TRAING is used to calculate the translation function. Finally FITING is used to refine the orientational and positional parameters of the molecule corresponding to the potential solutions, as a rigid body.

To carry out MR, the AMoRe package can be used. AMoRe constitutes a suite of programs written by Jorge Navaza (Navaza, 1993; Navaza, 1994). These

Automated molecular replacement package (AMoRe)

mounted on goniometer heads, which were in turn fixed on the oscillator dial of the image plate. However since our crystals suffered significant radiation damage at room temperature we decided to attempt cryo-crystallography and collected data at low temperature. Radiation damage to protein crystals is greatly reduced at lower than room temperatures (D. J. Haas, 1970; Low et al., 1966). Primary radiation damage is largely caused by interactions between the molecules in the crystal and the beam. This energy is dissipated in at least two ways; it produces thermal vibrations (heat) and it provides the necessary energy to break bonds between atoms in the molecules. Secondary damage to the crystals is caused by the diffusion of reactive radicals produced due to damage to the protein. This diffusion is aided by the presence of thermal energy. At cryo-temperature of around 1 OOK, thermal damage is limited and also the reactive products are immobilized and do not cause extensive secondary damage in areas of the crystal which are not exposed to the beam (Garman, 1999). For low temperature data collection, the crystals were initially soaked in a cryo-protectant, which was basically the mixture of the mother liquor and antifreeze. We added 30% glycerol to our mother liquor, in which the crystals were soaked from between 1 to 5 minutes to achieve cryo-protection. The crystals were then picked up using a 20Jl nylon loop, which was immediately flash frozen in a stream of nitrogen at 120k at a flow rate of 6 liters/min (Oxford cryo-systems). The crystals were centered in the beam using the two arcs and translations on the goniometer head and by viewing the crystal on the monitor of the attached CCD camera. The collimation, crystal to detector distance, oscillation angle and the exposure time per frame were optimized after a few trial frames in each case.

Data collection for macromolecular crystallography involves exposure of the crystal to X-rays and recording the intensities of the resultant diffraction patterns. Rapid advances in this field have made available sophisticated electronic detectors like the Image plate detector, high power X-ray generators and synchrotrons. Successful data set collection is followed by data processing to extract the hkl indices with corresponding intensities, along with an estimate of the errors involved. At the core of the Image Plate detector is an amorphous thin film made of Barium, Europium and Bromium. This material that is coated on to a motorized plate absorbs X-rays to form F-centers. These F-centers are the regions that store photon energy as excited electrons. After the exposure is complete the plate is read by a He-Ne (2eV) red laser. Absorption of photons induces excited electrons to return to ground state with the emission of blue light (4eV) which is quantitatively read by a photomultiplier. Exposing it to intense white radiation erases the plate. While the basic technology behind the image plates remains the same, improvements in electronics and computers has led to greater automation and faster data collection cycles. The X-ray intensity data for various Fab-peptide complexes of 36-65 were collected on the Mar345dtb, installed on a rotating anode X-ray source (RIGAKU, Japan) operating at 50kV and 1 OOmA (CuKa. radiation) with Osmic mirrors (RIGAKU, Japan). While the Mar225 image plate installed at BM14 (ESRF, Grenoble, France) was used to record three Fab-peptide complexes of BBE6.12H3. Data for antigen free BBE6.12 H3 Fab and its complex with Ppy peptide was recorded on Mar345dtb image plate (Mar research, Germany), installed on the home source. For data collection at room temperature, the crystals were mounted in 0.5 mm quartz capillary tube along with some mother liquor. The capillaries were then

X-ray intensity data collection

solution was injected into the HPLC. A salt gradient of 0 to 0.2 M NaCl over a period of 120 minutes was run and fractions for each peak, as detected by measurement of UV absorbance at 220nm, were collected. An aliquot of each fraction was subjected to acetone precipitation and the obtained precipitate was analyzed on SDS-PAGE to ascertain which fraction corresponds to IgG. The IgG fractions from different runs were pooled and concentrated to -1 mg/ml which was then dialyzed against the digestion buffer (0.15 M NaCl, O.lM Tris-Cl, pH 7.1).

The collected ascitic fluid was centrifuged to remove cell debris and fat. Mouse monoclonal ascites, was filtered through glass wool to remove lipid like material left over after centrifugation. The supernatant was then subjected to (Nr4)zS04 fractionation. Saturated (Nlit)zS04 solution (SAS) at pH 7.0 was gradually added to the ascites in an ice bath with continuous stirring till a concentration of 40% (v/v) was achieved. The mixture thus, obtained was centrifuged to get the protein pellet and the pellet was re-suspended in buffer (0.01 M Tris-Cl, pH 8.5). The crude antibody solution obtained from ammonium sulfate fractionation was dialyzed against the wash buffer (0.0 1 \1 Tris-Cl, pH 8.5) and then subjected to ion-exchange chromatography using 5PW-DEAE (60x150 mm) column on a Waters3000 preparative HPLC (Waters, L:SA), to purify IgG. All solutions used during chromatography were filtered (0.451-lm) and then degassed. Following equilibration of the column with wash buffer, a 2 ml aliquot of the crude antibody

Antibody purification

The peptides were purified using reverse-phase HPLC. Binding occurs through hydrophobic interactions between peptide and the column support. Decreasing the ionic nature or increasing the hydrophobicity of eluant such that it competes with peptide for hydrophobic groups on the column accomplishes elution. Crude peptides were purified on a Waters Xbridge™ BEH 130 reverse-phase C 18 column ( 19x250mm, 1 O~m, spherical) on a semi-preparative HPLC system (Waters, USA) using a linear gradient of 0.1% trifluoroacetic acid (Sigma) and acetonitrile (Merck). The absorption was monitored at 214nm. After purification, the peptides were lyophilized. The purity of the peptide was checked by determination of molecular mass using single quadruple mass analyzer (Fisons Instruments, UK). Circular dichroism studies were performed on the peptides to determine secondary structural state, if any, in solution. 50 )lM peptide concentrations in water were used and data was accumulated for 1 0 scans at a temperature of 10° C using a JASCO 710 spectropolarimeter. 1.0 nm bandwidth and O.lnm resolutions were used, with the sample being placed in a 2mm path length cuvette.

Peptide purification

All the peptides used in this study were synthesized by solid phase method on an automated peptide synthesizer (Applied Biosystems, Model 431A), using F-moc (9-fluorenylmethyloxycarbonyl) chemistry on a p-hydroxymethyl phenoxymethyl polystyrene resin (Nova Biochem). For the peptide synthesis, 0.1 mmol of the resin was used and deprotected using 20% piperidine in N-methyl-pyrrolidone (NMP). Subsequently 0.5nmol of the first amino acid was added and coupling was performed usmg DCC-HoBt (dicyclohexylcarbodiimide-hydroxybezotriazole) ester formation method. All other amino acids were coupled by DCC ester coupling. Amino acids and solutions required for peptide synthesis were procured from Nova Biochem and Applied Biosystems, respectively. After completion of synthesis, deprotection was carried out in 20% piperidine/DMF. Finally, the resin was shrunk using ether and dried under vacuum for a minimum of four hours. The cleavage was performed in dark using 94% TF A, 5% anisole, EDT and water accompanied by continuous stirring for two hours. The resin was then filtered and washed with DCM and the solution was evaporated on a rotary evaporator (Buchi, Switzerland) till only a small quantity of DCM/cleavage mixture is left. Cold anhydrous diethyl ether was added to the filtrate to aid in the separation of scavengers from the mixture. The peptides were then extracted with water using a separating funnel. Extraction was followed by evaporation of residual diethyl ether on the rotary evaporator. Total aqueous layer was then frozen as a thin film and lyophilized.

Procedure for peptide synthesis

the peptide and the resin is cleaved using trifluoroacetic acid (TF A) to release the polypeptide.

Solid phase peptide synthesis was introduced by Merrifield in 1963, and includes successive assembly of amino acid residues to build the peptide chain on an insoluble polymeric support. The C-terminal residue, with protected a-amino and side chain functional groups, is chemically attached to the insoluble resin via a flexible linker. Subsequently, in the coupling step, the a-amino group is deprotected and the next protected amino acid is reacted with the resin-bound first amino acid. This cycle of deprotection and coupling is repeated till the complete peptide chain is synthesized. After the synthesis of the desired peptide, the anchoring bond between

Peptide synthesis

Competent cells of the different strains of E. coli were prepared as described by Cohen et al. ( 1972). An LB-agar plate was streaked with the desired strain, and a single colony was inoculated into 5 ml of LB medium. The culture was grown at 37 °C with continuous shaking at 200 rpm for 6 hours. A small inoculum from this culture was used to start a I 00 ml culture in the same medium. At an OD600 of 0.3-0.4, when the culture reached early Jog phase, it was chilled on ice for 30 min .. and centrifuged at 2000 g for 15 min. at 4 °C. The pellet was gently resuspended in 50 ml of chilled 50 mM calcium chloride and incubated on ice for 60 min. The cell suspension was centrifuged at 2000 g for 15 min. at 4 °C, and the pellet was gently resuspended in 5 ml of chilled 50 mM calcium chloride containing 20% glycerol. The competent cell suspension was immediately aliquoted in prechilled vials and stored at -70 °C.

Preparation of Competent Bacterial Cells

Cancer cell lines of human origin, HUT102, T-cell leukemia; K562, erythroleukemia; COL0205; colon adenocarcinoma; MCF7, breast adenocarcinoma; A431, epidermoid carcinoma; A549, lung carcinoma and HeLa, cervical carcinoma and J774A.I, mouse monocyte-macrophage; and L929, mouse fibroblast were obtained from ATCC. All the cell Jines were maintained in RPMI 1640 supplemented with antibiotic antimycotic solution, 2 mM glutamine and I 0% heat inactivated foetal calf serum (Life Technologies, Maryland, USA). E. coli strain DH5a was used for DNA manipulation, cloning and mutagenesis. Strains CJ236 and DH5aF' were used. for oligonucleotide mediated site directed mutagenesis. BL21 (A.DE3) strain containing T7 RNA polymerase gene under the control of lac promoter, was used for expression of the recombinant proteins.

Cell Lines and Bacterial Strains

administered at two sites. In addition, the primary dose also contained 500 J..Lg of SPLPS as an additional adjuvant. This was followed by 2 booster at 4 weekly intervals with an equal amount of r-bZP3-DT conjugate.

Female New Zealand White rabbit (Small Animal Facility, National Institute of Immunology, New Delhi, India), 6 months of age was immunized intramuscularly with r-bZP3-DT conjugate equivalent to 125 Jlg of r-bZP3 (expressed in SG13009[pREP4] cells) in 0.9% saline emulsified with Squalene and Arlacel "A" in a ratio of 4: I and

Immunization of Rabbit

650C in 0.2X SSC, 0.1 % SDS for I 0 min. The membrane was wrapped in Saran wrap and exposed to an X-ray film. The colonies that were positive by colony hybridization were inoculated in a 3 ml culture and used for preparing DNA for analysis by restriction digestion and Southern blotting. The digested DNA was resolved on a 0.8% agarose gel as described above. The gel was soaked in 4 volumes of denaturing solution (1.5 M NaCI and 0.5 M NaOH) for 1 h at RT with shaking followed by neutralization (1 M Tris HCI, pH 8 and 1.5 M NaCI) for 1 hat RT. The DNA was transferred to a Nylon membrane, UV crosslinked and hybridized with the full length 32p labeled· bZP3 probe as described above.

The ligation mixture was used for transformation of DH5a cells as described earlier. Transformed bacterial colonies growing on LB Amp plates were screened by colony hybridization. Briefly, colonies were grown for 6-8 h on a Nylon membrane placed on a LB Amp plate. The colonies were lysed by placing the membrane on a Whatman® 3MM paper soaked in I 0% SDS for 3 min, followed by treatment with denaturing solution (0.5 N NaOH, I.5 M Nael) for 5 min and neutralization solution (0.5 M Tris Hel pH 8, 1.5 . M Nael) for 5 min in the same manner. The membrane was dried, UV cross linked (Ultraviolet crosslinker, Amersham) and processed for prehybridization and hybridization. Stocks of 20X sse (174 giL NaCI, 88.2 giL sodium citrate, pH 7.0) and 50X Denhardt's (I% ficoll, I% PVP, I% BSA) were prepared. The membrane was prehybridized for 4-6 h in the prehybridization solution (5X SSe, 5X Denhardt's, 0.5% SDS, I 0 J..Lg/ml sheared and denatured salmon sperm DNA). The bZP3 DNA was labelled using the Multiprime DNA labeling system using 50 ng of purified bZP3 DNA. For hybridization with the probe, I o6 cpm/ml of the denatured 32p labeled bZP3 probe was added to the prehybridization solution and incubation was further carried out for I4-I6 h. For removing the non specifically bound probe, the membrane was washed successively at RT in 2X sse for 10 min, at 55°e in 0.2X sse, 0.1% SDS for 10 min and finally at

Screening of the Recombinant Transfer Vector

Peptide antisera were generated in the laboratory against peptides PI, 23-45 aa residues with an extra lysine at the N-terminus (KQPFWLLQGGASRAETSVQPVL VE), P2, 300-322 aa residues (CSFSKSSNSWFPVEGPADICQCC) and P3, 324-347 aa residues (KGDCGTPSHSRRQPHVVSQWSRSA) corresponding to bZP3 precursor protein in rabbits and were used to determine their reactivity with the r-bZP3 protein expressed in E. coli in an enzyme linked immunosorbent assay (ELISA). Microtitration plates were coated with 200 ng of r-bZP3 or I J.tg/well of the peptide. HRPO conjugated goat anti-rabbit Ig at I :5000 dilution was used as revealing Ab.

Reactivity with Anti-peptide Sera

E. coli strains deficient in specific proteases were used to study their influence on the expression of r-bZP3. BL21 (DE3) and BL21 (pLysS) deficient in ompT and ion proteases and DF5 carrying a targeted mutation of the ptr gene, were transformed with the pQE-bZP3 plasmid. Colonies obtained were grown 0/N and subcultured next morning and grown till A6oo=0.7. Cultures were then induced with 0.5 mM IPTG for 3 h. Harvested cells were checked by SDS-P AGE and immunoblotting.

Expression of r-bZP3 in Different E. coli Strains

Conditions for expression of r-bZP3 in SG 13009[pREP4] cells transformed with the pQE-bZP3 plasmid were standardized. Cells were grown till A6oo=0.7 and induced with different concentrations of IPTG (0.5, 1, 2, or 4 mM) for a constant time period (3h) or induced with a 0.5 mM IPTG for different time periods (0, 1, 2, 3 or 5 h). Cells were harvested and analyzed by SDS-PAGE and immunoblot as described above.

Standardization of Expression Conditions

A I 00 ml culture was grown and induced according to the procedure mentioned above. The culture was divided into 2 aliquots and cells were pelleted down. For cytosolic localization, one pellet was resuspended in 5 ml of sonication buffer (50 mM Na-phosphate, pH 7.8, 300 mM NaCI). The sample was frozen and then thawed in ice-water and cells lysed by brief sonication. The sample was centrifuged at I 0,000 g for 20 min. The soup and the pellet represent the soluble and insoluble components of the cell pellet. In order to check for periplasmic localization, the 2nd aliquot of cells was resuspended in I 0 ml of hypertonic solution (30 mM Tris, pH 8, 20% sucrose, 1 mM EDT A) and incubated at RT for 10 min with shaking. Cells were centrifuged at 8,000 g for 10 min. The pellet was subjected to osmotic shock in 5 mM MgS04. Cells were stirred for 10 min in an ice water bath, centrifuged at 8000 g at 4°C for I 0 min. The soup collected represented the periplasmic fraction. The fractions were analyzed by 0.1% SDS-1 0% PAGE and Western blotting as described above.

Intracellular Localization

The cell pellet obtained from l ml culture was solubilized by boiling for 5 min in 100 J..Ll of 2X sample buffer (0.0625 M Tris, pH 6.8, 2% SDS, 10% glycerol, 5% BME and 0.001% bromophenol blue) and the proteins were resolved on a 0.1% SDS-10% PAGE (Laemmeli, 1970). The gel was stained with Coomassie brilliant blue for staining total cellular proteins. For immunoblotting, the proteins were electrophoretically transferred to 0.45 J..Lm nitrocellulose membrane overnight at a constant voltage of 15 V in Tris glycine buffer with 20% methanol (Towbin et al., 1979). Nonspecific sites on the membrane were blocked by incubation with 5% BSA in 50 mM phosphate buffered saline (PBS), pH 7 .4, for 1 h followed by 3 washes (15 min each) with PBS containing 0.1% Tween-20 (PBST). For detection of bZP3, a murine monoclonal antibody (MAb), MA-451, generated against the pZP3P and recognizing a cross reactive epitope (166-171 aa residues) within the bonnet sequence was used (Afzalpurkar and Gupta, 1997). The membrane was incubated for 1 h with a 1 :5 dilution of MA-451 culture supernatant, followed by 3 washes in PBST. Horseradish-peroxidase (HRPO) conjugated goat anti-mouse immunoglobulin (lg) was used to reveal bound Ab. Colour was developed with 0.6% (w/v) 4-chloronaphthol in 50 mM PBS, pH 7.4, containing 25% methanol and 0.06% H202. The reaction was stopped by washing the membrane with PBS.

SDS-PAGE and Immunoblot

The pQE-bZP3 plasmid was transformed in Ml5[pREP4] and SG13009[pREP4] bacterial strains provided with the kit. The transformed colonies were analyzed for expression. A single transformed colony was inoculated and grown overnight at 37oc in 1 ml of LB containing 100 J..Lg/ml of ampicillin and 25 J..Lg/ml of kanamycin. Cells were subcultured 1:10, next morning and grown until cell density reached an A600 of approximately 0.6-0.7. The cells were further grown in the presence of isopropyl P-D thiogalactopyranoside (IPTG) to induce expression of the fusion protein under the T -5 promoter. The cells were collected by centrifugation at 13,000 g for 60 sec and the resulting pellet was stored at -700C until used.

Expression in MJS[pREP4] and SG13009[pREP4] E. coli Strains

vector, under the phage T7 promoter, in BL21 (DE3) cells, and under the T5 phage promoter, in the pQE30 vector for expression in SG13009[pREP4] and M15[pREP4] cell strains. For cloning in pRSET B, the full length bZP3 initially subcloned in the pBacPAK8 vector at the Kpn I and Sac I sites was released after digestion with Kpn I and EcoR I and cloned in a similarly restricted pRSETB vector inframe with an N-terminal His6 tag. For cloning in the pQE30 vector, the pBacPAK8 carrying the full length bZP3 was initially digested with Not I, filled in with Klenow and then digested with Kpn I. The purified bZP3 fragment was then cloned in the vector digested with Kpn I and Sma I in frame with an N-terminal His6 tag. Though transformants positive for the bZP3 insert in the right reading frame were recovered, no expression could be detected by SDS-PAGE or immunoblots in either case. An alternate strategy was then devised in which an internal fragment of the gene, excluding the signal sequence and the transmembrane-like domain, following the putative furin cleavage site, was amplified by PCR using the forward primer 5'-CGGGATCCCAACCCTTCTGGCTCTTG-3' incorporating a BamH I site and the reverse primer 5'-CCGAGCTCAGAAGCAGACCTGGACCA-3' incorporating a Sac I site. The PCR was done in a 50 J!l volume using 50 pM of each primer and Vent polymerase for extension. The pBluescript-bZP3 (1 0 ng) having a full length bZP3 insert was used as the template and was initially denatured at 95°C for 10 min. Amplification was carried out for 35 cycles of denaturation at 95°C for 2 min, primer annealing at 600C for 2 min and extension at 72°C for 3 min followed by a final extension at 72oc .for 15 min. The amplified bZP3 fragment was digested with BamH I and Sac I and cloned in frame downstream of a His6 tag under the T5 promoter-lac operator control in the pQE30 vector. The authenticity of the construct was confirmed by N-terminal sequencing using an upstream sequencing primer GGCGT ATCACGAGGCCCTTTCG.

Our initial attempts to express the full length gene in E. coli as a His6 fusion protein failed. Attempts were initially made to express the His6-bZP3 protein in the pRSET B

PCR Amplification and Cloning in pQE30 Vector

CLONING AND EXPRESSION IN E. coli

ligation reactions were carried out usmg conditions and buffers specified by the manufacturer.

The PCR amplified eDNA fragment corresponding to bZP3 was resolved on a 0.8% agarose gel run using IX TAE buffer (0.04 M Tris-acetate, O.OOI M EDTA) and purified using the Geneclean® II kit. The PCR amplified bZP3 was digested with Kpn I and Sac I and ligated into the pBluescriptll SK(+) vector at the same sites. The digestion and

Agarose Gel Electrophoresis, Digestion and Ligation

The RIA for alpha hCG was similar to that used to estimate ~hCG. A monoclonal antibody specific to alpha hCG ( Gupta et al. , 1985 was used for the assay. The standard used was total hCG.

RIA for alpha hCG.

PBS and then replenished with the complete medium. Two days following transfection, the cells were subcultured into the appropriate selective medium for selection of stable clones as described below.

Calcium phosphate mediated stable transfections were performed by the method of Graham and Van der Eb ( 1973 with modifications as described by Gorman ( 1986 ). For each plasmid, two petri dishes each containing 0. 5 x 106 CHO-K1 cells were used, with 10 ug of cesium purified DNA for each transfection. A mock transfection which did not contain any DNA, was performed simultaneously as negative control. Precipitation of the DNA was done with great care to ensure the obtention of a fine, translucent precipitate rather than a dense and opaque precipitate. The calcium phosphate I DNA precipitate was added in 4 ml medium to the cells and the cells incubated for 3 hours at 37°C. At this stage, the cells were examined under the microscope and a fine precipitate appeared as small grains all over the cells. The cells were washed once with serum free medium and a glycerol shock given for 3 minutes at 37°C. The cells were washed twice again with

Using calcium phosphate.

Stable transfection was performed into CHO-K1 cells by the following procedures

stable transfection.