[Final] Purification Of B-Gal Formal Report

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  • Purification of -galactosidase from Escherichia coli

    Purification of -galactosidase from Escherichia coli ML308 by Differential Precipitation and Ion Exchange Chromatography*

    Andy P. Chand1, Taylor J. Clarke2, and Jaymie E. Oentoro3

    1From the Department of Molecular Biology Laurier University, Waterloo, ON N2L 3C5

    2From the Department of Biological Sciences Durham College, Oshawa, ON L1H 7K4

    3From the Department of Chemical Biology University of Ontario Institute of Technology, Oshawa, ON L1H 7K4

    *Running title: Purification of -galactosidase from Escherichia coli

    To whom correspondence should be addressed: Jaymie E. Oentoro, Department of Chemical Biology, University of Ontario Institute of Technology. 2000 Simcoe Street N., Oshawa, ON, CAN. Tel.: (905) 721-8668 ext. 3467; Fax: (905) 721-3304; E-mail: jaymie.oentoro@uoit.net

    Keywords: -galactosidase; Escherichia coli; enzyme purification; protein purification; differential precipitation; ion exchange chromatography; SDS-PAGE; Western blot

    Background: Potential uses of Escherichia coli derived -galactosidase in industry demonstrate a need for the pure protein Results: Through differential precipitation and ion exchange chromatography, -galactosidase is isolated Conclusion: -galactosidase can effectively be recovered from E. coli Significance: Efficient purification of -galactosidase is crucial for practical applications of the protein in biotechnology

    ABSTRACT -galactosidase is an enzyme with high

    potential for various biotechnological applications. The protein was purified from ML308 Escherichia coli through differential precipitation and size exclusion and ion exchange chromatography. Specific enzyme

    activity was monitored through hydrolysis of o-nitrophenyl--D-galactopyranoside (ONPG) and increased through subsequent purification steps. The entire purification sequence resulted in an overall yield of 1.67%. The purity of the final product was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis and indicated the presence of some impurities. -galactosidase was identified through a Western blot and its individual subunits were estimated to have a molecular weight of 125kDa. Results indicate that further optimization of the purification process is required to obtain an efficient yield.

    INTRODUCTION -galactosidase has been used ubiquitously

    in conjunction with Escherichia coli

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  • Purification of -galactosidase from Escherichia coli

    throughout history; it has been researched by Lester and Bonner1 who studied E. coli and the influence of carbon sources on the production of -galactosidase; by Craves, Steers, and Anfinsen2 investigating -galactosidase in greater detail, covering purification, composition, and molecular weight; and Loeffler, Sinnott, Sykes, and Withers3 focused on the interaction of -galactosidase with inhibitors, along with many others.

    -galactosidase has 3 main enzymatic functions; to cleave lactose into glucose and galactose (permitting entry to glycolysis) to catalyze the transgalactosylation of lactose to allolactose, and to cleave allolactose into monosaccharides4.

    The process of purifying -galactosidase from E. coli involves introducing the enzymatic active segment into the plasmid5. After the introduction and the quick replication rate of E. coli in appropriate culture and environmental conditions, the concentration of the enzyme for purification will increase as the culture grows. The cells can then undergo lysis to release the enzyme, which can then go through chromatographic processes to isolate the enzyme from other fragments from the cells. Once purified, the -galactosidase can be used to remove lactose from foods, can be used as a biomarker for detecting senescence/aging cells6, to produce galacto-oligosaccharides (GOS, prebiotics that provide a plethora of benefits to humans)7, and can be used in as a blue-white screen (a quick and easy way to detect of bacteria that have lost a specific plasmid)8.

    Lactose is the usual substrate for -galactosidase, but it can also bind with the nongalactose part of substrates, in fact many different substrates can be accepted9. One example includes X-gal, which is useful in blue-white screening, a process that allows for

    quick and easy detection of certain bacteria colonies in molecular cloning experiments. In order to activate, -galactosidase needs minerals to be fully active; these minerals include sodium, potassium, and magnesium10; the minerals play a role in binding and reactivity, but it has been discovered that even without these cations the enzyme has residual activity3.

    -galactosidase is an enzyme with several uses in biotechnology, from removing lactose to producing prebiotics with many health benefits to humans. Producing the enzyme can be very simple and quick with the help of introducing the enzyme into E. coli, which can lead to quick development with quick growth rates of the E. coli. The structure of -galactosidase allows it to interact with various active substrates, which allows it to carry out the various functions mentioned before.

    There have been other scientists who have researched E. coli and its role with -galactosidase. Kung, Spears, and Weissrach looked at synthesizing --galactosidase in-vitro by using E. coli ribosomes, a salt wash, and a supernatant fraction11. The results of the experiment showed that the synthesis of the protein is dependent on E. coli, its ribosomes, and the ribosomes dependencies to synthesize the compound of interest (L7 & L12)11. Fukuda looked at purifying and characterizing Endo--galactosidase from Esherichia freundii induced by hog gastric mucin. Degraded hog gastric mucin was used to induce the enzyme with culture medium, which would be purified with ammonium sulfate fractionation, DEAE-Sephadex c h r o m a t o g r a p h y , a n d a f f i n i t y chromatography12. This method is useful because mucin i s read i ly ava i lab le commercially and the efficiency of the enzyme production was high without significant induction. This paper reports a

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  • Purification of -galactosidase from Escherichia coli

    combination of purification techniques including differential precipitation, size exclusion chromatography, and ion exchange chromatography to purify -galactosidase.

    EXPERIMENTAL PROCEDURES Production of Escherichia coli Biomass To a loosely capped, sterile, plastic culture tube, minimal broth with lactose (4mL) was added. The broth was inoculated with ML308 (ATCC #15224) Strain E. coli and shaken at 200RPM and 37oC for approximately 24 hours before inoculating 500mL of minimal broth with the starter culture and being stored at 4oC.

    Harvesting Bacterial Cell By High-Speed Centrifugation The Allegra X-12R high-speed centrifuge was chilled to 4oC, prior to spinning any samples, with the appropriate SX4750 rotors and adapters. The 500mL starter culture was divided between 2 pre-weighted 250mL centrifuge bottles and then spun at 3750RPM for 20 minutes at 22oC to pellet the cells. After spinning, the supernatant was decanted back into the empty baffled flask to leave the bacterial pellet behind. Bacterial samples were kept on ice to minimize bacterial movement and dispersion of the pellet. The weight of the pellet was determined before storage at -20oC.

    Cell Lysate Preparation by Sonification Breaking buffer was prepared with the addition of 0.5M DTT to make a final concentration of 5mM (excess was stored and used for future solutions with the addition of fresh DTT). The bacterial pellet was then resuspended in breaking buffer by using 9mL (5mL per 1g of bacterial pellet). The suspension was then transferred to a 50mL centrifuge tube with the addition of a PMSF tablet for sonification. The sonification equipment was set up with ice to prevent overheating. The sample was swirled prior to sonification to resuspend the pellet, and then

    the sonification probe was inserted into the sample and turned on (70% maximum power in three 15 second increments with 30 second breaks to allow the sample to cool down). The homogenate was transferred to cold 1.5mL microcentrifuge tubes and then centrifuged for 10 minutes at 3750xg at 4oC. The centrifuged cell lysates were then combined in a Falcon tube, with 50L set aside for SDS-PAGE and Western Blotting. All samples were then stored at -20OC.

    Differential Precipitation Salting Out - Crystalline ammonium sulphate (2203.9mg) was added to thawed E. coli lysate (231mg per mL of cell lysate) over a period of 30 minutes on an orbital platform (sample was kept cold and gently mixed during the additions by placing the orbital shaker in the 4oC fridge). Approximately 2mL of 1M NaOH (1mL for every 10g of ammonium sulphate) was added to the cell lysate and mixed overnight. The sample was spun down in the pre-chilled centrifuge (4oC) at 10,000xg for 10 minutes. The supernatant was saved and stored at 4oC, while the precipitated protein was dissolved in breaking buffer and kept in 1.5mL microfuge tubes (50L was kept aside for SDS-PAGE and Western Blotting) then stored at 4oC.

    Desalting and Buffer Exchange by Size Exclusion Chromatography A 10cm long, 1.5cm diameter column was filled with a slurry of Sepharose G-75 beads and 0.2M NTM buffer to a 4 6cm bed height. The resin bed was equilibrated with 0.2M NTM buffer. Before loading the sample, it was centrifuged at full speed at 4oC for 5 minutes to pellet the non-soluble particles. The supernatant was run with 0.2M NTM buffer and 1mL fractions were collected and simultaneously assayed for -galactosidase activity.

    Purification of -galactosidase by Ion Exchange Chromatography DEAE-

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  • Purification of -galactosidase from Escherichia coli

    Sephadex resin was prepared by hydrating 0.