Discovery of active proteins directly from combinatorial randomized protein libraries without display, purification or sequencing:identification of novel zinc finger proteins.

We have successfully linked protein library screening directly with the identification of active proteins, without the need for individual purification, display technologies or physical linkage between the protein and its encoding sequence. By using 'MAX' randomization we have rapidly constructed 60 overlapping gene libraries that encode zinc finger proteins, randomized variously at the three principal DNA-contacting residues. Expression and screening of the libraries against five possible target DNA sequences generated data points covering a potential 40,000 individual interactions. Comparative analysis of the resulting data enabled direct identification of active proteins. Accuracy of this library analysis methodology was confirmed by both in vitro and in vivo analyses of identified proteins to yield novel zinc finger proteins that bind to their target sequences with high affinity, as indicated by low nanomolar apparent dissociation constants.

On-chip immunoprecipitation for protein purification

Immunoprecipitation (IP) is one of the most widely used and selective techniques for protein purification. Here, a miniaturised, polymer-supported immunoprecipitation (µIP) method for the on-chip purification of proteins from complex mixtures is described. A 4 µl PDMS column functionalised with covalently bound antibodies was created and all critical aspects of the µIP protocol (antibody immobilisation, blocking of potential non-specific adsorption sites, sample incubation and washing conditions) were assessed and optimised. The optimised µIP method was used to obtain purified fractions of affinity-tagged protein from a bacterial lysate.

Purification of plasmid DNA for use in human gene therapy

Two key issues defined the focus of this research in manufacturing plasmid DNA for use In human gene therapy. First, the processing of E.coli bacterial cells to effect the separation of therapeutic plasmid DNA from cellular debris and adventitious material. Second, the affinity purification of the plasmid DNA in a Simple one-stage process. The need arises when considering the concerns that have been recently voiced by the FDA concerning the scalability and reproducibility of the current manufacturing processes in meeting the quality criteria of purity, potency, efficacy, and safety for a recombinant drug substance for use in humans. To develop a preliminary purification procedure, an EFD cross-flow micro-filtration module was assessed for its ability to effect the 20-fold concentration, 6-time diafiltration, and final clarification of the plasmid DNA from the subsequent cell lysate that is derived from a 1 liter E.coli bacterial cell culture. Historically, the employment of cross-flow filtration modules within procedures for harvesting cells from bacterial cultures have failed to reach the required standards dictated by existing continuous centrifuge technologies, frequently resulting in the rapid blinding of the membrane with bacterial cells that substantially reduces the permeate flux. By challenging the EFD module, containing six helical wound tubular membranes promoting centrifugal instabilities known as Dean vortices, with distilled water between the Dean number's of 187Dn and 818Dn,and the transmembrane pressures (TMP) of 0 to 5 psi. The data demonstrated that the fluid dynamics significantly influenced the permeation rate, displaying a maximum at 227Dn (312 Imh) and minimum at 818Dn (130 Imh) for a transmembrane pressure of 1 psi. Numerical studies indicated that the initial increase and subsequent decrease resulted from a competition between the centrifugal and viscous forces that create the Dean vortices. At Dean numbers between 187Dn and 227Dn , the forces combine constructively to increase the apparent strength and influence of the Dean vortices. However, as the Dean number in increases above 227 On the centrifugal force dominates the viscous forces, compressing the Dean vortices into the membrane walls and reducing their influence on the radial transmembrane pressure i.e. the permeate flux reduced. When investigating the action of the Dean vortices in controlling tile fouling rate of E.coli bacterial cells, it was demonstrated that the optimum cross-flow rate at which to effect the concentration of a bacterial cell culture was 579Dn and 3 psi TMP, processing in excess of 400 Imh for 20 minutes (i.e., concentrating a 1L culture to 50 ml in 10 minutes at an average of 450 Imh). The data demonstrated that there was a conflict between the Dean number at which the shear rate could control the cell fouling, and the Dean number at which tile optimum flux enhancement was found. Hence, the internal geometry of the EFD module was shown to sub-optimal for this application. At 579Dn and 3 psi TMP, the 6-fold diafiltration was shown to occupy 3.6 minutes of process time, processing at an average flux of 400 Imh. Again, at 579Dn and 3 psi TMP the clarification of the plasmid from tile resulting freeze-thaw cell lysate was achieved at 120 Iml1, passing 83% (2,5 mg) of the plasmid DNA (6,3 ng μ-1 10.8 mg of genomic DNA (∼23,00 Obp, 36 ng μ-1 ), and 7.2 mg of cellular proteins (5-100 kDa, 21.4 ngμ-1 ) into the post-EFD process stream. Hence the EFD module was shown to be effective, achieving the desired objectives in approximately 25 minutes. On the basis of its ability to intercalate into low molecular weight dsDNA present in dilute cell lysates, and be electrophoresed through agarose, the fluorophore PicoGreen was selected for the development of a suitable dsDNA assay. It was assesseel for its accuracy, and reliability, In determining the concentration and identity of DNA present in samples that were eleclrophoresed through agarose gels. The signal emitted by intercalated PicoGreen was shown to be constant and linear, and that the mobility of the PicaGreen-DNA complex was not affected by the intercalation. Concerning the secondary purification procedure, various anion-exchange membranes were assessed for their ability to capture plasmid DNA from the post-EFD process stream. For a commercially available Sartorius Sartobind Q15 membrane, the reduction in the equilibriumbinding capacity for  ctDNA in buffer of increasing ionic demonstrated that DNA was being.adsorbed by electrostatic  interactions only. However, the problems associated with fluid distribution across the membrane demonstrated that the membrane housing was the predominant cause of the .erratic breakthrough curves. Consequently, this would need to be rectified before such a membrane could be integrated into the current system, or indeed be scaled beyond laboratory scale. However, when challenged with the process material, the data showed that considerable quantities of protein (1150 μg) were adsorbed preferentially to the plasmid DNA (44 μg). This was also shown for derived Pall Gelman UltraBind US450 membranes that had been functionalised by varying molecular weight poly-L~lysine and polyethyleneimine ligands. Hence the anion-exchange membranes were shown to be ineffective in capturing plasmid DNA from the process stream. Finally, work was performed to integrate a sequence-specific DNA·binding protein into a single-stage DNA chromatography, isolating plasmid DNA from E.coli cells whilst minimising the contamination from genomic DNA and cellular protein. Preliminary work demonstrated that the fusion protein was capable of isolating pUC19 DNA into which the recognition sequence for the fusion-protein had been inserted (pTS DNA) when in the presence of the conditioned process material. Althougth the pTS recognition sequence differs from native pUC19 sequences by only 2 bp, the fusion protein was shown to act as a highly selective affinity ligand for pTS DNA alone. Subsequently, the scale of the process was scaled 25-fold and positioned directly following the EFD system. In conclusion, the integration of the EFD micro-filtration system and zinc-finger affinity purification technique resulted in the capture of approximately 1 mg of plasmid DNA was purified from 1L of E.coli  culture in a simple two stage process, resulting in the complete removal of genomic DNA and 96.7% of cellular protein in less than 1 hour of process time.

The ecology of percolating filters containing a plastic filter medium in relation to their efficiency in The treatment of domestic sewage

The suitability of a new plastic supporting medium for biofiltration was tested over a three year period. Tests were carried out on the stability, surface properties, mechanical strength, and dimensions of the medium. There was no evidence to suggest that the medium was deficient in any of these respects. The specific surface (320m2m-3) and the voidage (94%) of the new medium are unlike any other used in bio-filtration and a pilot plant containing two filters was built to observe its effects on ecology and performance. Performance was estimated by chemical analysis and ecology studied by film examination and fauna counts. A system of removable sampling baskets was designed to enable samples to be obtained from two intermediate depths of filter. One of the major operating problems of percolating filters is excessive accumulation of film. The amount of film is influenced by hydraulic and organic load and each filter was run at a different loading. One was operated at 1.2m3m-3day-1 (DOD load 0.24kgm-3day-1) judged at the time to be the lowest filtration rate to offer advantages over conventional media. The other filter was operated at more than twice this loading (2.4m3m-3day-lBOD load 0.55kgm-3day-1) giving a roughly 2.5x and 6x the conventional loadings recommended for a Royal Commission effluent. The amount of film in each filter was normally low (0.05-3kgm(3 as volatile solids) and did not affect efficiency. The evidence collected during the study indicated that the ecology of the filters was normal when compared with the data obtained from the literature relating to filters with mineral media. There were indications that full ecological stability was yet to be reached and this was affecting the efficiency of the filters. The lower rate filter produced an average 87% BOD removal giving a consistent Royal Commission effluent during the summer months. The higher rate filter produced a mean 83% BOD removal but at no stage a consistent Royal Commission effluent. From the data on ecology and performance the filters resembled conventional filters rather than high rate filters.

Purification and characterisation of two cancer cachexia factors

Cachexia is a wasting phenomenon that often accompanies malignant disease. Its manifestation is associated with shortened survival and reduced responsiveness to anti-tumour therapy and as yet there is no established, effective amelioratory treatment. The MAC 16 model of cancer cachexia has been shown by many studies to closely mirror the human condition. Thus, cachexia is mediated by the presence of a small, slow-growing solid tumour that is mainly resistant to chemotherapy. In addition, the condition is largely attributable to aberrations in metabolic processes, while weight loss due to anorexia is negligible. Cachexia induced by the MAC 16 tumour, has been shown to be mediated by the production of tumour-derived circulatory catabolic factors, and the further elucidation of the structure of these molecules contributes towards the main content of this report. Thus, a factor with in vitro lipid-mobilising activity has been purified from the MAC 16 tumour, and has been found to have similarities to tumour-derived lipolytic factors published to date. Further work demonstrated that this factor was also purifiable from the urine of a patient with pancreatic cancer, and that it was capable of inducing weight loss in non tumour-bearing mice. Sequence analysis of the homogeneous material revealed an identity to Zn-α-2-glycoprotein, the significance of which is discussed. An additional factor, first detected as a result of its specific reactivity with a monoclonal antibody produced by fusion of splenocytes from MAC 16 tumour-bearing mice with mouse BALB/c myeloma cells, was identified as a co-purificant during studies to isolate the lipolytic factor. Subsequent purification of this material to homogeneity resulted in the determination of 18 of the N-terminal amino acids and revealed the highly glycosylated nature of its structure. Thus, this material (P24) was found to have an apparent molecular mass of 24kD of which 2kD was due to protein, while the remainder (92%) was due to the presence of carbohydrate groups. Sequence analysis of the protein core of P24 revealed an identity with Streptococcal pre-absorbing antigen (PA-Ag) in 11 of the amino acids, and the significance of this is discussed. P24 was shown to induce muscle protein breakdown in vitro and to induce cachexia in vivo, as measured by the depletion of fat (29%) and muscle (14%) tissue in the absence of a reduction of food and water intake. Further studies revealed that the same material was purifiable from the urine of patients with pancreatic cancer and was found to be detectable in the urine of cancer patients with weight loss greater than l.Skg/month. Thus, cachexia induced by the MAC 16 tumour in mice and by malignant disease in humans may be induced by similar mediators. Attempts to isolate the gene for P24 using information provided by the N-terminal protein sequence were unsuccessful. This was probably due to the low abundance o[ the material, as determined by protein purification studies; and the nature of the amino acids of the N-terminal sequence, which conferred a high degree o[ degeneracy to the oligonucleotides designed for the polymerase chain reaction.

Enterotoxigenic Escherichia coli infection of pigs: expression, extraction, purification and the adhesive properties of the K88 fimbrial adhesin

The ability of Escherichia coli to express the K88 fimbrial adhesin was satisfactorily indicated by the combined techniques of ELISA, haemagglutination and latex agglutination. Detection of expression by electron microscopy and the ability to metabolize raffinose were unsuitable. Quantitative expression of the K88 adhesin was determined by ELISA. Expression was found to vary according to the E.coli strain examined, media type and form. In general it was found that the total amount was greater, while the amount/cfu was less on agar than in broth cultures. Expression of the K88 adhesin during unshaken batch culture was related to the growth rate and was maximal during late logarithmic to early stationary phase. A combination of heat extraction, ammonium sulphate and isoelectric precipitation was found suitable for both large and small scale preparation of purified K88ab adhesin. Extraction of the K88 adhesin was sensitive to pH and it was postulated that this may affect the site of colonisation of by ETEC in vivo. Results of haemagglutination experiments were consistent with the hypothesis that the K88 receptor present on erythrocytes is composed of two elements, one responsible for the binding of K88ab and K88ac and a second responsible for the binding of the K88ad adhesin. Comparison of the haemagglutinating properties of cell-free and cell-bound K88 adhesin revealed some differences probably indicating a minor conformational change in the K88 adhesin on its isolation. The K88ab adhesin was found to bind to erythrocytes over a wide pH range (PH 4-9) and was inhibited by αK88ab and αK88b antisera. Inhibition of haemagglutination was noted with crude heparin, mannan and porcine gastric mucin, chondrosine and several hexosamines, glucosamine in particular. The most potent inhibitor of haemagglutination was n-dodecyl-β-D-glucopyranoside, one of a series of glucosides found to have inhibitory properties. Correlation between hydrophobicity of glucosides tested and degree of inhibition observed suggested hydrophobic forces were important in the interaction of the K88 adhesin with its receptor. The results of Scatchard and Hill plots indicated that binding of the K88ab adhesin to porcine enterocytes in the majority of cases is a two-step, three component system. The first K88 receptor (or site) had a K2. of 1.59x1014M-1 and a minimum of 4.3x104 sites/enterocyte. The second receptor (or site) had a K2 of 4.2x1012M-1 with a calculated 1.75x105 sites/enterocyte. Attempts to inhibit binding of cell-free K88 adhesin to porcine enterocytes by lectins were unsuccessful. However, several carbohydrates including trehalose, lactulose, galactose 1→4 mannopyranoside, chondrosine, galactosamine, stachyose and mannan were inhibitory. The most potent inhibitor was found to be porcine gastric mucin. Inhibition observed with n-octyl-α-D-glucopyranose was difficult to interpret in isolation because of interference with the assay, however, it agreed with the results of haemagglutination inhibition experiments.

The effect of catchment characteristics on sewage settling velocity grading

A procedure has been developed which measures the settling velocity distribution of particles within a complete sewage sample. The development of the test method included observations of particle and liquid interaction using both synthetic media and sewage. Comparison studies with two other currently used settling velocity test procedures was undertaken. The method is suitable for use with either DWF or storm sewage. Information relating to the catchment characteristics of 35 No. wastewater treatment works was collected from the privatised water companies in England and Wales. 29 No. of these sites were used in an experimental programme to determine the settling velocity grading of 33 No. sewage samples. The collected data were analysed in an attempt to relate the settling velocity distribution to the characteristics of the contributing catchment. Statistical analysis of the catchment data and the measured settling velocity distributions was undertaken. A curve fitting exercise using an S-shaped curve which had the same physical characteristics as the settling velocity distributions was performed. None of these analyses found evidence that the settling velocity distribution of sewage had a significant relationship with the chosen catchment characteristics. The regression equations produced from the statistical analysis cannot be used to assist in the design of separation devices. However, a grading curve envelope was produced, the limits of which were clearly defined for the measured data set. There was no evidence of a relationship between settling velocity grading and the characteristics of the contributing catchment, particularly the catchment area. The present empirical approach to settling tank design cannot be improved upon at present by considering the variation in catchment parameters. This study has provided a basis for future research into the settling velocity measurement and should be of benefit to future workers within this field.

Chemical constituents associated with sewage settling velocity profiles

A methodology has been developed to measure the chemical constituents associated with the settling velocity fractions that comprise a wastewater settling velocity profile (SVP). 31 wastewater samples were collected from fifteen different catchments in England and Wales. For each catchment, settling velocity and associated chemical constituent profiles were determined. The results are mainly for Suspended Solids (SS), Chemical Oxygen Demand (COD), Phosphorus (P) and Total Kjeadahl Nitrogen (TKN), however these are supplemented by the results from 5 events for a suite of heavy metals. COD, P, Hg, Mn and Pb were found to be predominantly associated with the solid phase and TKN, Al, Cu and Fe with the liquor phase of the wastewater samples. The results in the thesis are expressed as mass of pollutant (g) per mass total SS (kg). COD and P were found to be mainly associated with the sinkers and had a particular affinity for solids with settling velocities in the range 0.9-9.03mm/sec. TKN was mainly associated with the soluble phase, however of the solids that did settle, a peak was found to be associated within the settling velocity range 0.9-9.03mm/sec. The relationships identified for COD and P were generally found to be unaffected by flow conditions and catchment characteristics. However, TKN was found to be affected by catchment type. Data on the distribution of heavy metals was limited, and no specific relationships with solids were identified. 16 mean pollutant profiles are presented in the thesis. Presentation of the data in this form will enable the results to be of use in the design of sedimentation devices to predict removal efficiencies for solids and associated pollutants. The findings of the research may also be applied to modelling tools to provide further characteristics on the solids that are modelled than is currently used. This would enhance the overall performance of tools used in integrated catchment modelling.

Surfactant-free purification of membrane proteins with intact native membrane environment

In order to study the structure and function of a protein, it is generally required that the protein in question is purified away from all others. For soluble proteins, this process is greatly aided by the lack of any restriction on the free and independent diffusion of individual protein particles in three dimensions. This is not the case for membrane proteins, as the membrane itself forms a continuum that joins the proteins within the membrane with one another. It is therefore essential that the membrane is disrupted in order to allow separation and hence purification of membrane proteins. In the present review, we examine recent advances in the methods employed to separate membrane proteins before purification. These approaches move away from solubilization methods based on the use of small surfactants, which have been shown to suffer from significant practical problems. Instead, the present review focuses on methods that stem from the field of nanotechnology and use a range of reagents that fragment the membrane into nanometre-scale particles containing the protein complete with the local membrane environment. In particular, we examine a method employing the amphipathic polymer poly(styrene-co-maleic acid), which is able to reversibly encapsulate the membrane protein in a 10 nm disc-like structure ideally suited to purification and further biochemical study.

Dual affinity method for plasmid DNA purification in aqueous two-phase systems

The DNA binding fusion protein, LacI-His6-GFP, together with the conjugate PEG-IDA-Cu(II) (10 kDa) was evaluated as a dual affinity system for the pUC19 plasmid extraction from an alkaline bacterial cell lysate in poly(ethylene glycol) (PEG)/dextran (DEX) aqueous two-phase systems (ATPS). In a PEG 600-DEX 40 ATPS containing 0.273 nmol of LacI fusion protein and 0.14% (w/w) of the functionalised PEG-IDA-Cu(II), more than 72% of the plasmid DNA partitioned to the PEG phase, without RNA or genomic DNA contamination as evaluated by agarose gel electrophoresis. In a second extraction stage, the elution of pDNA from the LacI binding complex proved difficult using either dextran or phosphate buffer as second phase, though more than 75% of the overall protein was removed in both systems. A maximum recovery of approximately 27% of the pCU19 plasmid was achieved using the PEG-dextran system as a second extraction system, with 80-90% of pDNA partitioning to the bottom phase. This represents about 7.4 microg of pDNA extracted per 1 mL of pUC19 desalted lysate.

Characteristics of the upper phase of bio-oil obtained from co-pyrolysis of sewage sludge with wood, rapeseed and straw

Sewage sludge was pyrolysed with 40% mixed wood, 40% rapeseed and 40% straw. The reason for the mixture of different biomass is to investigate the impact of co-pyrolysis on the upper phase of bio-oil in terms of changes to composition, elemental analysis, viscosity, water content, pH, higher heating value and acid number that could impact on their applications. The biomass was pyrolysed in a laboratory at 450 °C and bio-oil was collected from two cooling traps. The bio-oil obtained from co-pyrolysis of sewage sludge with wood, rapeseed and straw was analysed for composition using the gas chromatography mass spectrometry. The upper phase from the co-pyrolysis process was also characterised for ultimate analysis, higher heating values, water content, viscosity, pH and acid number. There was an increase in the amount of upper phase produced with co-pyrolysis of 40% rapeseed. It was also found that the upper phase from sewage sludge with mixed wood has the highest viscosity, acid number and lowest pH. The bio-oil containing 40% straw was found to have a pH of 6.5 with a very low acid number while the 40% rapeseed was found to have no acid number. Sewage sludge with 40% rapeseed was found to have the highest energy content of 34.8 MJ/kg, 40% straw has 32.5 MJ/kg while the 40% mixed wood pyrolysis oil has the lowest energy content of 31.3 MJ/kg. The 40% rapeseed fraction was found to have the highest water content of 8.2% compared to other fractions.