[Final] Purification Of B-Gal Formal Report

Purification of β-galactosidase from Escherichia coli

Purification of β-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 β-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: [email protected]

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

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

ABSTRACT β-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-β-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. β-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 β-galactosidase has been used ubiquitously

in conjunction with Escherichia coli

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throughout history; it has been researched by Lester and Bonner1 who studied E. coli and the influence of carbon sources on the production of β-galactosidase; by Craves, Steers, and Anfinsen2 investigating β-galactosidase in greater detail, covering purification, composition, and molecular weight; and Loeffler, Sinnott, Sykes, and Withers3 focused on the interaction of β-galactosidase with inhibitors, along with many others. β-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 β-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 β-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 β-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, β-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. β-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 β-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 β-galactosidase. Kung, Spears, and Weissrach looked at synthesizing Β--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-β-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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combination of purification techniques including differential precipitation, size exclusion chromatography, and ion exchange chromatography to purify β-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 50µL 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 (50µL 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 β-galactosidase activity.

Purification of β-galactosidase by Ion Exchange Chromatography – DEAE-

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Sephadex resin was prepared by hydrating 0.3g of DEAE-Sephadex resin with 0.2M NTM Buffer and then packed into a 10cm long, 1.5cm diameter column. 0.2M NTM buffer was used to equilibrate the column until the pH of eluted buffer was equivalent to the pH of the fresh buffer. The sample was loaded onto the column and the eluent was collected in a UV-transparent cuvette. The column was ran until the absorbance reading of the eluent dropped below 0.100, using 0.2M NTM buffer as the blank.

Protein elution was carried out by way of 10mL concentration steps using 0.2M, 0.3M, 0.4M and 0.5M NTM buffer. 1mL fractions were collected from this elution process and each fraction was qualitatively assayed for β-galactosidase by adding 50µL of the fraction and 1mL of Z buffer to 20µL of ONPG solution. 100µL of the fractions showing the greatest β-galactosidase activity were stored at 4oC.

Preparation of SDS-PAGE gels – Two S D S - PA G E g e l s w e r e p r e p a r e d t o concentrations of 8.5%. Once polymerized, the combs were left in the gels and the gels were placed in layers of wet paper towel and wrapped in cling wrap. The gels were then stored in a plastic container at 4oC.

SDS-PAGE & Western Blot Analyses of Reserved Fractions – Unstained size standards were prepared for both the Coomassie Blue stained gel and the gel used for Western blotting by diluting 5µl of unstained protein standards with 5µl of roH2O and 10µl of 2x sample buffer and then the standards were treating by boiling them. Additionally, a pre-stained size standard was prepared for Western blotting by diluting 5µL of the pre-stained protein standard with 7.5µL of 2x sample buffer and 7.5µL of roH2O. Reserved fractions (15µL) were loaded on each gel, alongside the prepared standards. Samples were run at a

voltage of 106V for approximately an hour. I m m e d i a t e l y u p o n c o m p l e t i o n o f electrophoresis, the gel intended for Western blotting was electrophoretically transferred to a nitrocellulose membrane. The membrane was stored in plastic cling wrap at 4oC. The other gel was left to stain in Coomassie Blue for one week.

Western Blotting and Coomassie Blue Staining of SDS-PAGE gels – A Western blot was performed on nitrocellulose membrane using a series of washes in blocking solution and an hour incubation in both primary and secondary antibodies. Prior to incubation in the secondary antibodies, 2ml of 1x alkaline phosphatase reagent was added to the nitrocellulose membrane to produce a colour reaction. The nitrocellulose membrane was then photographed and annotated once a brown colour developed. The Coomassie Blue stained gel was destained by pouring the staining solution back into the original stock bottle. The gel was then destained by submerging the gel in destaining solution and placing it on a shaker until the gel was fully destained. The destained gel was then photographed and annotated.

Enzyme and Protein Assays - Bradford13 and β-galactosidase14 assays were conducted at weeks 2, 3, 5, and 6 to determine protein concentration and β-galactosidase activity. The Bradford assay was conducted with standards of bovine serum albumin at 12.5, 25, 50, and 100µg in breaking buffer and samples volumes of 50, 10, and 5µL. A595

readings were recorded to measure protein concentration. β-galactosidase assays involved sample

preparation at volumes of 50, 10, 5, and 1µL diluted in breaking buffer to obtain a final volume of 50µL, and then the addition of 1mL of Z-buffer. The samples were incubated in a water bath (37oC) for a few minutes before the

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addition of ONPG solution; the samples were then incubated at 37oC for approximately 5 minutes until a “banana-yellow” colour appeared. A420 readings were recorded to measure β-galactosidase activity. One unit of activity is defined as the amount of enzyme liberating one µmol of ONPG per minute at 37oC.

RESULTS Purification of β-galactosidase - A 1.8g

pellet was harvested from the E. coli biomass. After sonification, 9ml of cell lysate was recovered and its contents established as maximum potential yields; total protein weight of 20.74mg and 2.42 units of β-galactosidase (Table 1). Salting out with ammonium sulphate precipitated 2.58mg of protein out of solution, including 1.91 units of β-galactosidase (Table 1). The supernatant contained 0.0248 units of β-galactosidase, a 1.03% loss of desired protein (Table 1).

Size exclusion chromatography desalted the protein mixture and exchanged its buffer with 0.2M NTM buffer, culminating in three 1ml fractions containing a total of 1.86 units of β-galactosidase. The highest purification factor during this experiment was achieved at this step with a value of 6.08x (Table 1). Following ion exchange purification, 28mls in separate 1mL fractions were collected and verified to possess β-galactosidase activity. The amount of protein dropped dramatically at this step and consequently was undetectable by the Bradford assay due to unreliable absorbance values (Table 1). However, β-galactosidase activity continued to be apparent with 0.00402 units at a total percent yield of 1.7% (Table 1). The entirety of the purification process is summarized in Table 1.

SDS-PAGE of Reserved Samples and Purified β-galactosidase - Samples taken after each purification step (not including size

exclusion) were separated on an 8.5% polyacrylamide SDS gel and stained with Coomassie blue to generate Figure 2. Size exclusion samples were not saved for SDS-PAGE since it was all used during the final purification step. Although the same volume of sample was included in each lane, they had variable amounts of protein (cell lysate 31.1µg; precipitated protein 38.7µg; and an unknown amount of ion exchange purified β-galactosidase). Despite variable amounts of protein, the separated samples indicate an effective purification of β-galactosidase since the number of protein bands between subsequent purification steps decreases (Fig. 2).

In the pellet and lysate separations (Fig. 2, Lanes 3 and 4) there is tremendous smearing present while there is none found in the purified β-galactosidase sample. The smeared lanes also have a large amount of protein bands, of which, the smaller sized ones (Fig. 2, Lanes 3 and 4, Bands K - N and U - X) do not appear in the purified β-galactosidase sample. The 5 bands present in the sample of purified β-galactosidase have molecular weights of 152, 76, 50, 40, and 32kDa in order of increasing migration distance (Fig. 2, Lane 2).

The most intense bands of each sample occur towards the top of the gel, of which, the most apparent one in purified β-galactosidase is at a migration distance outside of the scope of the size standard (Fig. 2, Lane 2, Band A). Consequently, the extrapolated molecular weight of 152kDa will be inaccurate.

Western Blotting of Reserved Samples and Purified β-galactosidase - The same samples analyzed in SDS-PAGE were probed for β-galactosidase with the use of anti-β-galactosidase and a colour reaction to generate Figure 3. Because of the high protein amount used in each sample, the bands appear

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smeared and have a lot of overlap which can contribute to inaccuracies (Fig. 3). Uncoloured amorphous shapes are scattered throughout the membrane and potentially obscured a possible colour reaction (Fig. 3). Despite this abnormality, a positive colour change occurred in all samples analyzed; lysate, the prec ip i ta ted pe l le t , and pur i f ied β -galactosidase (Fig. 3). Another oddity is the pair of bands that cell lysate and precipitated protein both display with apparently high molecular weights outside the range of the standard curve (Fig. 3, Lane 2 and 3, A and E).

The strongest intensity is present in the sample of precipitated protein with a molecular weight of approximately 122kDa (Fig. 3, Lane 3, B and F). Cell lysate also has an intense band around the same molecular weight (Fig. 3, Lane 2, Band B). Nevertheless, these bands are incredibly smeared and are likely inaccurate. However, purified β-galactosidase has a band at a molecular weight of about 125kDa (Fig. 3, Lane 4, Band G). Continuing with intensity, lysate protein bands are visibly lighter than those of the precipitate, and purified β-galactosidase bands are even lighter that those (Fig. 3).

DISCUSSION The purification procedure followed in the

present study demonstrates an efficient isolation of purified active β-galactosidase resulting in a final yield of 1.67% of the crude enzyme activity, comparable to other studies investigating purification of this particular enzyme15. As the purification progressed, the presence of a positively increasing specific activity trend (Fig. 1) is consistent with expected reports from Cohn16. Also congruent with the same study16 is the decrease of total protein amount, further supporting the purification of desired enzyme (Fig. 1 and Table 1).

The 79.1% yield of β-galactosidase from differential precipitation is comparable and efficient in comparison to other studies which have recovered between the ranges of 10% - 77.2% after similar precipitation methods15,17. During this step, a minimal amount, 1.03%, of the enzyme of interest was left behind in the supernatant, indicated by the coloured bands of the Western blot (Fig 3., Lane 3) and the specific activity of the sample (Table 1). This loss is efficient when compared to other studies who have reported losses of more than 50% of desired protein in their crude extract15. To further enhance this process and decrease the amount of protein loss via decanting, a greater volume of supernatant could be left with the pellet so as to not accidentally remove the desired protein.

During ion exchange chromatography, a drastic loss of desired enzyme occurred, potentially due to improper column stripping. In future attempts, the column should be run for a greater amount of time to ensure collection of all remaining protein. However, even though the total protein amount was undetectable via Bradford assay, it still retained functionality (Table 1). Thus, it can be inferred that of the remaining protein, a large percentage of it, if not all, is pure β-galactosidase. In place of ion exchange, affinity exchange chromatography could be done to enhance specificity and selection of β-galactosidase. It has been reported by Pollard et al. that using agarose as a support and p-aminophenylfl-n-thiogalactopyranoside as an ligand for β-galactosidase results in an effective purification18.

The SDS-PAGE gels did not run to completion and resulted in poor resolution which may affect the results obtained. Furthermore, the cell lysate and precipitated protein samples were too concentrated and subsequently cause inaccuracies due to

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imprecise migration distances (Fig.2, Lanes 2 and 3). The excessive smearing of this sample is likely due to the presence of concentrated ammonium sulphate salts as Dickson et al. reported similar results19. These high protein concentrations and presence of salt may have also affected the size standard and purified β-galactosidase since their bands appear to be warped in the direction of the aforementioned samples (Fig. 2, Lanes 1 and 4). These potential sources of error could be solved by performing another gel with lower and equal sample concentrations.

Since cell lysate is a crude extract, multiple proteins were apparent in its electrophoresed sample, some of which decreased in intensity following precipitation (Fig. 2, Lane 2 and 3, Bands K - N and U - X). The presence of these bands still remaining in precipitated protein sample indicates that an amount of supernatant was left with the pellet. This was intentional in order to ensure minimal loss of product as mentioned before.

These undesired proteins were removed after ion exchange purification since the sample no longer has these bands (Fig. 2, Lane 1). Furthermore, the bands are more resolved due to less salt and a suitable protein concentration. Based on standard curve calculations these proteins have sizes of roughly 152, 76, 50, 40, and 32kDa (Fig. 2, Lane 1). It is likely that these values are inaccurate due to aforementioned reasons. However, the 152kDa band is likely due the subunit of the desired protein of interest, β-galactosidase, which is known to be a 464kDa homotetramer20. The smaller protein bands are likely other residual proteins, indicating impurity presence in the purification process. They may also potentially be degraded

subunits of β-galactosidase which were susceptible to proteases.

Despite the inaccuracies from the SDS PAGE gel carrying over to the Western blot, the most prominent bands of the samples correspond to the molecular weight of β-galactosidase (Fig. 3, Lane 2, 3, and 4, Bands B, F, and G) which is the heaviest band in the size standard lane. While the lysate and precipitate fractions are subject to estimation due to smearing, the purified β-galactosidase has a definitive band at a molecular weight of about 125kDa complete with colouration (Fig. 3, Lane 4, Band G). The colour change along with the similarity in molecular weight suggest that this is purified β-galactosidase. Since the smaller protein bands visible in the gel for the purified fraction do not register a colour change, it can be inferred that these are contaminant proteins. In order to remove these undesired proteins, the purification process can be modified to include other purification techniques such as affinity chromatography or gel purification.

Th i s s tudy demons t r a t ed tha t β -galactosidase, isolated from E. coli ML308 can be purified effectively. For a greater yield, care should be taken while doing the ion exchange purification to ensure complete recovery of all β-galactosidase. Increasing the purity of the yield obtained can be achieved when multiple purification techniques are used in con junc t ion such as d i ffe ren t ia l p r e c i p i t a t i o n a n d i o n e x c h a n g e chromatography. Further studies are required to optimize the recovery of a good yield of β-galactosidase.

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REFERENCES

1. Lester, G. and Bonner, D.M. (1952) The Occurrence Of Beta-Galactosidase In Escherichia coli. Osborn Botanical Laboratories. 63, 759 – 769.

2. Craven, G. R., Steers, E. Jr., and Anfinsen, C. B. (1964) Purification, Composition, and Molecular Weight Of The Beta-Galactosidase Of Escherichia coli. Journal Of Biological Chemistry. 240, 2468 – 2477.

3. Loeffler, R. S., Sinnot, M. L., Sykes, B. D., and Withers, S. G. (1979) Interaction Of Beta-Galactosidase Of Escherichia coli With Some Beta-D-Galactopyranoside Competitive Inhibitors. Biochem. J. 177, 145 – 152.

4. Juers, D. H., Matthews, B. W., and Huber, R. E. (2012) LacZ Beta-Galactosidase: Structure And Function Of An Enzyme Of Historical And Molecular Biological Importance. Protein Sci. 21, 1792 – 1807.

5. Casadaban, M. J., Chou, J., and Cohen, S. N. (1980) In Vitro Gene Fusions That Join An Enzymatically Active Beta-Galactosidase Segment To Amino Terminal Fragments Of Exogenous Protein: Escherichia coli Plasmid Vectors For The Detection And Cloning Of Translational Initiation Signals. Journal Of Bacteriology. 143, 971 – 980.

6. Lee, B. Y., Han, J. A., Im, J. S., Morrone, A., Johung, K., Goodwin, E. C., Kleijer, W. J., DiMaio, D., and Hwang, E. S. (2006) Senescence-Associated β-Galactosidase Is Lysosomal β-Galactosidase. Aging Cell. 5, 187–195.

7. Asraf, S. S. and Gunasekaran, P. (2010) Current Trends Of β-Galactosidase Research And Application. Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology. 2, 880 - 890.

8. Arnaud, M., Chastanet A., and Debarbouille, M. (2004) New Vector For Efficient Allelic Replacement In Naturally Nontransformable, Low-GC-Content, Gram-Positive Bacteria. Appl. Environ. Microbiol. 70, 6887 - 6891.

9. Egal, R. (1979) The Lac-Operon For Lactose Degradation, Or Rather For The Utilization Of Galactosylglycerols From Galactolipids. J. Theor. Biol. 79, 117 – 119.

10. Cohn, M. and Monod, J. (1951) Purification And Properties Of Beta-Galactosidase (Lactase) In Escherichia coli. Biochem. Biophys. Acta. 7, 153 – 174.

11. Kung, H.F., Spears, C., and Weissrach, H. (1974) Purification And Properties Of A Soluble Factor Required For The Deoxyribonucleic Acid-Directed In-Vitro Synthesis Of Beta-Galactosidase. Journal Of Biological Chemistry. 250, 1556 – 1562.

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12. Fukuda, M. N. (1980) Purification And Characterization Of Endo-Beta-Galactosidase From Escherichia freundii Induced By Hog Gastric Mucin. Journal Of Biological Chemistry. 256, 3900 – 3905.

13. Bradford, M. M. (1970) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 72, 248 – 254.

14. Hung, M. N. and Lee, B. H. (1998) Cloning and expression of β-galactosidase gene from Bifidobacterium infantis into Escherichia coli. Biotechnol Lett. 20, 659 - 662.

15. Jagota, S. K., Ramana, M. V. and Dutta, S. M. (1981) Beta-Galactosidase of Streptococcus cremoris H. Journal of Food Science. 46, 161 - 168.

16. Cohn, M. (1957) Contributions of studies on the beta-galactosidase of Escherichia coli to our understanding of enzyme synthesis. Bacteriological Reviews. 3, 140 - 68.

17. Hung, M. N. and Lee, B. H. (2002) Purification and characterization of a recombinant β-galactosidase with transgalactosylation activity from Bifidobacterium infantis HL96. Apple Microbiol Biotechnol. 58, 439 - 445.

18. Pollard, H. B., Cuatrecasas, P., and Steers, E. (1971) The Purification of β-Galactosidase from Escherichia coli by Affinity Chromatography. Journal of Biological Chemistry. 246, 196 - 200.

19. Dickson, R. C., Dickson, L. R., and Markin, J. S. (1979) Purification and Properties of an Inducible, B-Galactosidase Isolated from the Yeast Kluyveromyces lactis. Journal of Bacteriology. 137, 51 - 61.

20. Jacobson, R. H., Zhang, X. J., Dubose, R. F., and Matthews, B. W. (1994) Three-dimensional structure of β-galactosidase from E. Coli. Nature. 369, 761 – 766.

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Table 1. β-galactosidase Purification Data Summary

StepEnzyme Activity

(units/ml)

Protein Concentration

(mg/ml)

Total Volume

(ml)Yield

(units)

Total Mass (mg)

Specific Activity

(units/mg)

Percent Yield (%)

Purification Factor (x)

Lysate 0.268 2.07 9.0 2.42 18.7 0.129 100 1

(NH4)2SO4 Precipitation Supernatant

0.00248 1.52 10.0 0.0248 15.2 0.00164 1.03 0.013

(NH4)2SO4 Precipitation

Pellet1.91 2.58 1.0 1.91 2.58 0.740 79.1 5.72

Size Chrom. 0.619 0.787 3.0 1.86 2.36 0.787 76.9 6.08

Ion Chrom. 0.00144 - 28.0 0.0402 - - 1.67 -

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1 - E. coli Cell Lysis 2 - (NH4)2SO4 Precipitation 3 - Size Exclusion Chromatography 4 - Ion Exchange Chromatography Total protein refers to the percentage ratio of the protein mass obtained at that purification step to the protein mass initially obtained at cell lysis. Desired protein refers to the percentage ratio of β-galactosidase units obtained at that purification step to the β-galactosidase units initially obtained at cell lysis. Purification factor for each step is the ratio of β-galactosidase units to protein mass.

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0

5

10

15

20

0

25

50

75

100

1 2 3 4

Prot

ein

(%)

Purification Step

Figure 1. Purification of β-galactosidase

Total Protein Desired Protein "Purification Factor"


Figure 2. β-galactosidase Samples Purified from ML308 Escherichia coli on Coomassie

Blue Stained 8.5% SDS-PAGE

1 - Purified β-galactosidase after Ion Exchange Chromatography 2 - (NH4)2SO4 Precipitated Protein 3 - E. coli Cell Lysate 4 - Protein Size Standards

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1 2 3 4

X

Q

VW

UTSR

P

MLK

JI

H

G

F

EDC

B

A O 116

66

4535

25

18

Size in kDa

N


Figure 3. Western Blot of β-galactosidase Samples Purified from Escherichia coli by 8.5% SDS-PAGE on Nitrocellulose

Membrane After Reaction with Alkaline Phosphatase

1 - Pre-stained Protein Size Standards 2 - E. coli Cell Lysate 3 - (NH4)2SO4 Precipitated Protein 4 - Purified β-galactosidase after Ion Exchange Chromatography

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1 2 3 4

20

120

26

86

47

34

Size in kDa

C

D

A

GB F

E


Table 2. Standard Curve Migration Distance and Sizes for 8.5% SDS-PAGE

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Known Size (kDa) Log of Size Measured Migration (mm) Log of Migration

18 1.26 471 2.67

25 1.40 372 2.57

35 1.54 268 2.43

45 1.65 197 2.29

66 1.82 115 2.06

116 2.06 50 1.70


Example Calculation

B - With a migration of 173mm;

log (173) = 2.24

From the standard curve y ≃ 1.70

101.70 ≃ 50

∴ The fragment is approximately 50 kDa.

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Figure 4. Standard Curve for Protein Molecular Weight Determination

Log

of P

rote

in M

olec

ular

Wei

ghts

(kD

a)

1.25

1.5

1.75

2

2.25

Log of Protein Migrations (mm)1.6 1.9 2.2 2.5 2.8


Table 5. Calculated Sizes of Figure 2 - Lane 4: Ion Exchange Fractions

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Band Lysate Measured Migration (mm)

Log of Migration

Corresponding Log of Size

Interpolated Size (kDa)

A 24 1.38 2.18 152

B 99 2.00 1.88 76

C 173 2.24 1.70 50

D 223 2.35 1.60 40

E 278 2.44 1.51 32


Table 6. Calculated Sizes of Figure 2 - Lane 3: Differential Precipitation Pellet Band Lysate Measured

Migration (mm)Log of Migration Corresponding Log

of SizeInterpolated Size

(kDa)

F 32 1.51 2.14 138

G 103 2.01 1.87 74

H 194 2.29 1.65 45

I 251 2.40 1.55 36

J 315 2.50 1.46 29

K 389 2.59 1.36 23

L 425 2.63 1.32 21

M 465 2.67 1.28 19

N 503 2.70 1.24 17

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Table 7. Calculated Sizes from Migration Distances of Figure 2 - Lane 2: Cell Lysate

Band Lysate Measured Migration (mm) Log of Migration Corresponding Log


(kDa)

O 36 1.56 2.12 132

P 68 1.83 1.98 96

Q 109 2.04 1.85 71

R 191 2.28 1.66 46

S 241 2.38 1.57 37

T 314 2.50 1.46 29

U 376 2.58 1.38 24

V 417 2.62 1.33 21

W 461 2.66 1.28 19

X 493 2.69 1.25 18

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Table 8. Standard Curve Migration Distance and Sizes for Western Blot

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Known Size (kDa) Log of Size Measured Migration (mm) Log of Migration

120 2.08 131 2.12

86 1.93 208 2.32

47 1.67 333 2.52

34 1.53 385 2.59

26 1.41 580 2.76

20 1.30 658 2.82


Example Calculation

With a migration of 137mm;

log (137) = 2.14

From the standard curve y ≃ 2.08

102.08 ≃ 120

∴ The fragment is approximately 120 kDa.

"20

Figure 5. Standard Curve for Protein Molecular Weight Determination of Western Blot

Log

of P

rote

in M

olec

ular

Wei

ghts

(kD

a)

1.39

1.58

1.77

1.96

2.15

Log of Protein Migrations (mm)2.05 2.275 2.5 2.725 2.95


Table 9. Calculated Sizes of Figure 3 - Lane 2: Cell Lysate Band Lysate Measured



(kDa)

A 43 1.63 2.55 351

B 137 2.14 2.08 120

C 223 2.35 1.86 72

D 278 2.44 1.75 56

"21


Table 10. Calculated Sizes of Figure 3 - Lane 3: Differential Precipitation PelletBand Lysate Measured



(kDa)

E 50 1.70 2.49 309

F 135 2.13 2.09 122

"22


Table 11. Calculated Sizes of Figure 3 - Lane 4: Purified β-galactosidaseBand Lysate Measured



(kDa)

G 132 2.12 2.10 125

"23


Table 12. Standard BSA Absorbance Readings and Cell Lysate Data Volume of 2μg/μL BSA

Added (μL)Mass of BSA (μg) A595

6.25 12.5 0.792

12.50 25 0.980

25.00 50 1.741

50.00 100 2.380

"24


Table 13. Cell Lysate A595 Readings and Data

Incubation time for all samples and Standards was 15 min 04 secs. *Absorbance value > 1.9; unreliable for analysis

Sample Added (μL)

Absorbances Calculated Mass (μg)

Concentration (μg/μL)

Average Concentration (μg/μL)

5 0.729 6.708 1.342

2.07410 1.124 28.059 2.806

50 1.919* - -

"25


"26

Figure 6. Standard BSA Absorbance Curve - Cell Lysate

Abs

orba

nce

at 5

95 n

m

0.92

1.34

1.76

2.18

2.6

Mass of BSA (µg)0 22 44 66 88 110


Table 14. Standard BSA Absorbance (NH4)2SO4 Pellet and Supernatant Readings and Data Volume of 2μg/μL BSA

Added (μL) Mass of BSA (μg) A595

6.25 12.5 0.409

12.50 25 0.662

25.00 50 0.992

50.00 100 1.189

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Table 15. (NH4)2SO4 Pellet and Supernatant Readings and Data

Incubation time for all samples and Standards was 17 min 28 secs. *Too low to obtain accurate info from standard curve

Sample Sample Added (μL) A595

Calculated Mass (μg)



Supernatant

5 0.259* - -

1.51710 0.525 12.583 1.258

50 1.165 88.774 1.775

Pellet

5 0.485 7.821 1.564

2.58010 0.752 39.607 3.961

50 1.350 110.798 2.216

"28


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Figure 7. Standard BSA Absorbance Curve - Differential Precipitation

Abs

orba

nce

at 5

95 n

m

0.44

0.68

0.92

1.16

1.4

Mass of BSA (µg)0 22 44 66 88 110


Table 16. Standard BSA Absorbance Readings and Data: Size Exclusion Chromatography Volume of 2μg/μL BSA


6.25 12.5 0.155

12.50 25 0.466

25.00 50 0.617

50.00 100 1.182

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Table 17. (NH4)2SO4 Pellet and Supernatant Readings and Data

Incubation time for all samples and Standards was 17 min 28 secs. *Absorbance value < 0.1; unreliable for analysis

Sample Added (μL) A595




5 0.049* - -

0.78710 0.101 0.855 0.085

50 0.910 74.400 1.488

"31


"

Graph 8. Standard BSA Absorbance CurveA

bsor

banc

e at

595

nm

0.3

0.55

0.8

1.05

1.3

Mass of BSA (µg)0 22 44 66 88 110

"32


Table 18. Standard BSA Absorbance Readings and Data - Ion Exclusion of FractionsVolume of 2μg/μL BSA


6.25 12.5 0.918

12.50 25 1.177

25.00 50 1.246

50.00 100 1.315

"33


Table 19. Ion Exclusion of Fractions Readings and Data

Incubation time for all samples and Standards was 17 min 45 secs. *Absorbance value < 0.1; unreliable for analysis

Sample Added (μL) A595




5 0.007* - -

-10 0.021* - -

50 0.068* - -

"34

[Final] Purification Of B-Gal Formal Report

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