Protein Purification

Identification of components present in biological complexes requires their purification to near homogeneity. Methods of purification vary from protein to protein, making it impossible to design a general purification strategy valid for all cases. We have devel-oped the tandem affinity purification (TAP) method as a tool that allows rapid purification under native conditions of complexes, even when expressed at their natural level. Prior knowledge of complex composition or function is not required. The TAP method requires fusion of the TAP tag, either N- or C-terminally, to the target protein of interest. Starting from a relatively small number of cells, active macromolecular complexes can be isolated and used for multiple applications. Variations of the method to specifi-cally purify complexes containing two given components or to subtract undesired complexes can easily be implemented. The TAP method was initially developed in yeast but can be successfully

Immobilized metal-ion affinity chromatography (IMAC) has been developed for the rapid isolation and purification of recombinant proteins. In this chapter, megaporous cryogels were synthesized having metal-ion affinity functionality, and their adsorptive properties were investigated. These cryogels have large pore sizes ranging from 10 to 100 μm with corresponding porosities between 80 and 90 %. The synthesized IMAC-cryogel had a total ligand density of 770 μmol/g. Twelve milligram of a His 6-tagged protein (NAD (P)H-dependent 2-cyclohexen-1-one-reductase) can be purified from a crude cell extract per gram of IMAC-cryogels. The protein binding capacity is increased with higher degrees of grafting, although a slight decrease in column efficiency may result. This chapter provides methodologies for a rapid single-step purification of recombinant His 6-tagged proteins from crude cell extracts using IMAC-cryogels.

This paper describes a suitable method for the optimum extraction and isolation of phycocyanin from the cyanobacterium Synechococcus sp. IO9201 isolated from Caribbean waters. Phycocyanin from this microorganism was purified to homogeneity and some of its properties were investigated. The purification steps consisted of extraction, hydrophobic interaction chromatography and ion exchange chromatography. Freezing at −21°C-thawing at 4°C, using an alkaline buffer was the best method for extracting phycocyanin from Synechococcus sp. IO9201. The best extraction was obtained using butyl-sepharose resin for hydrophobic interaction chromatography and 0.05 M Tris-HCl (pH = 7) containing 10% ethanol for phycocyanin elution. Finally, phycocyanin was further purified by ion exchange chromatography using Q-sepharose and eluted with a complex isocratic system. The estimated molecular weight of the phycocyanin purified from Synechococcus sp. IO9201 was 102 000 daltons by gel filtration and the isoelectric point was 4.6. When analyzed by SDS-PAGE, Synechococcus sp. IO9201 phycocyanin migrated as two bands having an apparent molecular weight of 21 360 and 18 980 Da. The first band corresponds to β phycocyanin subunits, whereas the second corresponds to α phycocyanin subunits. So, this phycocyanin was characterized as (αCPCβCPC)3.

There have been many attempts to understand the mechanisms that determine the activity and stability of enzymes at extreme temperatures. Previous studies often compared enzymes from mesophiles and thermophiles and speculated that the observed differences were factors involved in adaptation to a certain temperature range. Most of these studies concluded that gross structural changes such as the quantity of certain amino acids or the total number of hydrogen bonds are responsible for the adaptation of optimal activity to temperature extremes. However, these studies have compared similar enzymes from distantly related organisms leaving one to wonder if the differences noted are from an adaptation to temperature or merely the result of genetic drift.
In this dissertation, I present an analysis of the cold-active beta-galactosidase (BgaS) from the Arthrobacter sp. SB. A study of the primary sequence and modeled structure of BgaS showed that many of the proposed adaptations for optimal activity in the cold did not hold true for this enzyme. Consequently, I decided to alter the BgaS enzyme to determine the contributions of specific amino acids to activity and stability at low temperatures.
I first examined the area of BgaS that aligned with the domain five mobile loop of the LacZ beta-galactosidase of Escherichia coli. In LacZ this area (residues 794-803) aids the binding of substrate and alterations at residue 794 increased the catalytic efficiency of the enzyme with lactose. However, when similar mutations were made in bgaS, they caused either a complete loss or a decrease of activity showing that although this area is also important for BgaS function the alterations affect the enzymes differently.
To further explore the low temperature activity of BgaS, I screened for second site revertants of a null mutant resulting from a G803D change. Restoration of activity was accomplished with the addition of only two mutations (E229D and V405A). Separation of these two mutations into a wild type background yielded an enzyme with a three fold increase in catalytic efficiency with little effect on the thermostability. This shows that small and subtle changes to the enzyme can further increase the activity at low temperatures.
I also explored the thermostability of BgaS through directed mutagenesis and directed evolution studies. The rational mutagenesis targeted the C-terminal portion of BgaS, an area in LacZ known to affect thermostability. From this study, I discovered that a single cysteine to glycine or cysteine to serine mutation resulted in an increase in thermal optimum for BgaS and in another closely related enzyme. The directed evolution study primarily targeted the active site in an attempt to create a more active version of BgaS. One mutant obtained from this screen, with a single alteration in amino acid sequence, created an enzyme with more activity and a 3.3 fold increase in the time it remained active at 30C, compared to BgaS.
Through the combination of mutational analysis and biochemical characterization, I have shown that the introduction of a limited number of amino acid changes are sufficient to alter the activity and/or thermal properties of an enzyme whereas previous studies have suggested that multiple alterations would be required. I have also increased the thermal optimum of three closely related enzymes by altering one amino acid. Considering the large size of the beta-galactosidase subunit, the finding that one change in the C-terminal 25 residues has any effect on the enzyme, not to mention an up to 20C increase in temperature optimum, is quite interesting. Taken as a whole, this work illustrates that small, unpredicted changes in the amino acid sequence of even large enzymes can have dramatic effects on their thermostability and/or activity.

Protein denaturation in beche -de- mer stored at low temperatures was studied. The selected temperatures were 4 C, 0 C and – 10 C and durations of treatment were 12, 24, 48 and 72 hours. Decline in solubility of protein in 5% neutral salt (NaCl) solution was used as an index of denaturation. Protein denaturation increased with the duration of storage possibly due to uncoiling of polypeptides and cross linking among the adjacent chains of these complex molecules. Differences were noticed in the pattern of decline in the protein solubility at different temperatures. Of the various thermal regimes tested, chilling at –10 C appeared to be the most effective means of controlling protein denaturation.

In this study the potentiality of Bangladesh zircon as a raw material for opacifier was examined. Preparation of zircon flour was done by both wet and dry ball milling. Wet milling of zircon was found to be much efficient than dry milling. Zircon flour of peak size 8 μm obtained by wet milling was purified by leaching in concentrated H2SO4 acid. Leaching time was varied from 2 to 20 h and sulphuric acid concentration ranged from 10 to 80 ml/l. Both un-purified and purified Bangladesh zircon flour were tested for their performance as opacifier in a couple of local ceramic factories. The whiteness and glossiness of glazes made with Bangladesh zircon were compared with that made with imported zircon. It has been demonstrated that Bangladesh zircon is quite satisfactory for the production of zircon flour in the ceramic industries. Zircon flour made from Bangladesh zircon can effectively replace imported zircon flour in ceramic glaze, if adequate size reduction and purification is carried out.

A comparative study on weak anion exchangers was performed to investigate the pH dependence, binding strength, particle size distribution, and static and dynamic capacity of the chromatographic resins. The resins tested included: DEAE Sepharose FF, Poros 50 D, Fractogel EMD DEAE (M), MacroPrep DEAE Support, DEAE Ceramic HyperD 20, and Toyopearl DEAE 650 M. Testing was performed with five different model proteins: Anti-FVII mAb (immunoglobulin G), aprotinin, bovine serum albumin (BSA), Lipolase (Novozymes), and myoglobin. Retention showed an expected increasing trend as a function of pH for proteins with low pI. A decrease in retention was observed for some resins at pH 9 likely due to initiation of deprotonation of the weak anion-exchange ligands. Expected particle size distribution was obtained for all resins compared to previous studies. Binding strength to weak anion-exchange resins as a function of ionic strength depends on the specific protein. Binding and elution at low salt concentration may be performed with Toyopearl DEAE 650 M, while binding and elution at high salt concentration may be performed with MacroPrep DEAE Support. Highest binding capacities were generally obtained with Poros 50 D followed by DEAE Ceramic HyperD 20. A general good agreement was obtained between this study and data obtained by the suppliers. Verification of binding strength trends with model proteins was achieved with human growth hormone (hGH) and a hGH variant on the same resins with different elution salts, sodium chloride, sodium hydrogenphosphate, sodium sulphate, and sodium acetate. Static capacity measurements obtained in the traditional experimental set-up were compared with high-throughput screening (HTS) technique experiments with reasonable agreement. Isotherm data obtained from HTS techniques and pulse experiments were successfully combined with mathematical modelling to simulate, develop and optimise the separation process of two model proteins, Lipolase and BSA. The data presented in this paper may be used for selection of resins for testing in process development.

Phytochemical research has gained a lot of momentum in the medical field for the discovery of new, safe, and effective remedies. In the context of antimicrobial research, various plant sources have been discovered with the potential to disrupt bacterial quorum sensing (QS), which plays a key role in the regulation of virulence in many Gram positive and Gram negative bacteria. Pseudomonas aeruginosa, a Gram negative bacterium, is known to produce multiple QS systems that control the expression of virulence determinants and biofilm development in this pathogen. Hence, the inhibition of QS has been pursued as a promising therapy for treatment of drug resistant Pseudomonas infections. A comprehensive review of the research data available for plant products as QS inhibitors of the organism has been presented here, while further suggesting the future prospects for these inhibitors.

Pseudomonas putida is a saprotrophic soil bacterium that is able to degrade organic solvent and is useful in bioremediation. The aim of the study was to isolate metagenomic DNA of considerable purity and to study the diversity of Pseudomonas putida from different soil sample sites at the University of the Western Cape.
The DNA extracted was used for PCR amplification of a fragment of the Pseudomonas putida, following the successful agarose gel analysis. A nested-PCR strategy was employed using the universal primers (E9F and 1510R) which were combined with Pseudomonas putida specific primer (PA-GS-F and PA-GS-R). The ligation reaction mixture was used in the transformation of E.coli KRX, the transformation products were plated on Luria broth agar plates containing 100 µg/µl ampicillin. Results indicated that the 1:3 and 1:5 plates had only one isolated colony present which more were expected as there was enough insert for each vector. Colonies were present on the control plate which was not expected. The nested-PCR was used to overcome the problem of having too little DNA for use as PCR templates and the second round amplification produced a band of approximately 700bp.
Diversity of Pseudomonas putida was determined by using the amplicon of the correct size which was subjected to restriction endonuclease digestion (RsaI) and were viewed on 2% agarose gel electrophoresis.
Although the study was successful in determining the diversity of Pseudomonas putida it is clear that another study will have to be done in the future to ensure we have the overall diversity to produce conclusive results, as our study was done with less soil sample so it does not represent the overall population of Pseudomonas putida in the soil of the University of the Western Cape.

Background Affinity chromatography is one of the most efficient protein purification strategies. This technique comprises a one-step procedure with a purification level in the order of several thousand-fold, adaptable for various proteins, differentiated in their size, shape, charge, and other properties. The aim of this work was to verify the possibility of applying affinity chromatography in bacteriophage purification, with the perspective of therapeutic purposes. T4 is a large, icosahedral phage that may serve as an efficient display platform for foreign peptides or proteins. Here we propose a new method of T4 phage purification by affinity chromatography after its modification with affinity tags (GST and Histag) by in vivo phage display. As any permanent introduction of extraneous DNA into a phage genome is strongly unfavourable for medical purposes, integration of foreign motifs with the phage genome was not applied. The phage was propagated in bacteria expressing fusions of the phage protein Hoc with affinity tags from bacterial plasmids, independently from the phage expression system. Results Elution profiles of phages modified with the specific affinity motifs (compared to non-specific phages) document their binding to the affinity resins and effective elution with standard competitive agents. Non-specific binding was also observed, but was 102-105 times weaker than the specific one. GST-modified bacteriophages were also effectively released from glutathione Sepharose by proteolytic cleavage. The possibility of proteolytic release was designed at the stage of expression vector construction. Decrease in LPS content in phage preparations was dependent on the washing intensity; intensive washing resulted in preparations of 11-40 EU/ml. Conclusions Affinity tags can be successfully incorporated into the T4 phage capsid by the in vivo phage display technique and they strongly elevate bacteriophage affinity to a specific resin. Affinity chromatography can be considered as a new phage purification method, appropriate for further investigations and development.

The proximate composition of Bryophyllum pinnatum leaves was evaluated in both dry and fresh samples to determine the ash, carbohydrate, fat, fibre, protein, moisture and mineral contents using the method of Association of Analytical Chemist (A OAC) and Atomic Absorption Spectrophotometer (AAS).The result shows that Bryophyllum pinnatum contain (1.21 ± 0.07 and 0.8 ± 0.03%) ash, (72.92 ± 1.08 and 4.46 ± 0.52%) carbohydrate, (1.38 ± 0.06 and 1. 15 ± 0.05%) fat, (6.02 ± 1. 06 and 0.95 ± 0.06%) fibre, (5. 38 ± 0.10 and 1.61 ± 0.02%) protein, and (13.01 ± 1.03 and 91.03± 0.55%) moisture in dry and fresh samples respectively. The result of the mineral content revealed that Bryophyllum pinnatum is good sources of minerals such as ‘Fe, Zn, Cu, Mg, Na, K, Ca, Ni, Cd and Pd. This shows that Bryophyllum pinnatum leaf is a good source of human nutrition and should be included as dietary supplement.

Coagulant proteins from Moringa oleifera (MO) seeds were successfully purified by removing seed oil followed by a protein salting out method at 40% (NH 4) 2 SO 4 combined with subsequent dialysis and heat treatment. A microtiter plate-based coagulation activity assay was then performed using colloidal bentonite solution and alum as positive control. The results show an oil recovery of 40 ± 2% while the water-soluble protein fraction in the defatted MO seed was 12.5 ± 0.5 mg/g or 4.4% of the total seed protein content. Heating the desalted protein fraction to 121 • C helped to obtain a 94% pure protein of approximately 7 kDa. Computational analyses support the hypothesis that the coagulating MO protein is an intrinsically disordered protein, providing reason for its stability at extreme temperatures (121 • C). The purified, thermo-stable coagulating protein retained its coagulating activity paving the way to economically produce a sterile natural coagulant from MO seeds.

Carbapenems (imipenem, meropenem, biapenem, ertapenem, and doripenem)
are β-lactam antimicrobial agents. Because carbapenems have the broadest spectra among
all β-lactams and are primarily used to treat infections by multi-resistant Gram-negative
bacteria, the emergence and spread of carbapenemases became a major public health concern.
Carbapenemases are the most versatile family of β-lactamases that are able to hydrolyze
carbapenems and many other β-lactams. According to the dependency of divalent cations
for enzyme activation, carbapenemases can be divided into metallo-carbapenemases
(zinc-dependent class B) and non-metallo-carbapenemases (zinc-independent classes
A, C, and D). Many studies have provided various carbapenemase structures. Here
we present a comprehensive and systematic review of three-dimensional structures of
carbapenemase-carbapenem complexes as well as those of carbapenemases. We update
recent studies in understanding the enzymatic mechanism of each class of carbapenemase,
and summarize structural insights about regions and residues that are important in acquiring
the carbapenemase activity.

The emergence of antibiotic resistance has
increased the interest for finding new antimicrobials in the
past decade. Probiotic Lactic acid bacteria producing antimicrobial
proteins like bacteriocin can be excellent agents
for development as novel therapeutic agents and complement
to conventional antibiotic therapy. Uropathogenic
Escherichia coli, most causative agent of Urinary tract
infection, has developed resistance to various antibiotics. In
the present investigation, antibacterial substance like bacteriocin
(Bacteriocin DT24) produced by probiotic Lactobacillus
brevis DT24 from vaginal sample of healthy Indian
woman was partially purified and characterized. It was
efficiently working against various pathogens, that is, Uropathogenic
E. coli, Enterococcus faecium, Enterococcus
faecalis and Staphylococcus aureus. The antimicrobial
peptide was relatively heat resistant and also active over a
broad range of pH 2–10. It has been partially purified by
ammonium sulfate precipitation and gel filtration chromatography
and checked on reverse-phase high-performance
liquid chromatography. Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis of bacteriocin DT24 was
approximately 7-kDa protein. The peptide is inactivated by
proteolytic enzymes, trypsin and lipase but not when treated
with catalase, a-amylase and pepsin. It showed bacteriostatic
mode of action against uropathogenic E. coli. Such characteristics
indicate that this bacteriocin-producing probiotic
may be a potential candidate for alternative agents to control
urinary tract infections and other pathogens.

Enzyme-cleaved antibodies are used widely for the treatment of envenoming. Such products should comprise only ‘highly pure’ immunoglobulin fragments since Fc or other contaminating protein fragments or their aggregates may lead to side effects. The digestion of ovine antiserum and its purified IgG were investigated using pepsin and trypsin. Trypsin was effective at digesting purified IgG but unsuitable for the direct digestion of serum. In contrast, pepsin was highly effective at digesting all unwanted serum components to low molecular weight (≤13 kDa) fragments while leaving the ~100-kDa F(ab′)2 intact. The optimum pH for pepsin digestion was between 3.25 and 3.50. The effects of salt concentration and pH on the digestion products were investigated by size exclusion chromatography under various conditions, which revealed a pH-dependent aggregation of some of the low molecular weight Fc and non-IgG fragments. These high molecular weight aggregates were not shown by SDS-PAGE. Unwanted low molecular weight fragments could be removed simply by diafiltration with a 30-kDa nominal molecular weight cutoff membrane and piperazine buffer (containing 150 mM NaCl, pH 6), leaving an F(ab′)2 solution contaminated only with some pepsin and a small amount of the aggregated low molecular weight fragments. These highly acidic contaminants were then removed easily using an anion exchange column and the F(ab′)2 produced following a subsequent concentration step was essentially free from pepsin and aggregates with a purity of over 96% and a yield of 19.3 g F(ab′)2/l serum. This novel, high yield method for processing serum to highly pure F(ab′)2 avoids salt precipitation and centrifugation and should be suitable for large-scale production.

A rare γ-pyranopyrazole skeleton: design, one-pot synthesis and computational study †
Drawing upon a consecutive amide coupling and intramolecular cyclisation pathway, a one-pot, straightforward synthetic route has been developed for a range of pyrazole fused γ-pyrone derivatives. The reaction mechanism proposed for the chemoselective formation of γ-pyranopyrazole is furthermore fully supported by experimental and computational studies.

This project presents the process design of the extraction of good quality protein from cheap, commercially available rapeseed meal using food grade table salt and tap water at room temperature. The demand, the supply and growing opportunities of product application have been studied to determine the plant capacity. Laboratory scale production and testing of samples for crude protein content has been done using Micro Kjldahl apparatus. The process employed included a counter current spray drying. Finally the economic analysis has been dealt with briefly.