The effect of salts and chaperone proteins on Alkaline Phosphatase

stability

Aka: the results of 271 continuous enzyme activity assays

Mary KleinDhruve RingwalaStephanie Korte

Spring 2011Biochem 463A

Background• During the purification process of AP we heated the

lysate to 80° C for 15 minutes to remove all of the proteins not as thermally stable

• Kurokawa et al, in 2000, showed that E. coli over expression of protein disulfide isomerase DsbC stabilized proteins with multiple disulfide bonds in the periplasm, including AP

• During the purification, after running a column with a MgCl2 buffer, compared to the MgSO4 buffer used for dialysis, enzyme activity was lost

• Poe et al, in 1993, showed that NaCl lowered the activity of AP when compared to Na2SO4

Goals

• We want to discover if there is a protein in the periplasm of E. coli that leads to alkaline phosphatase’s high thermal stability and, if that protein is lost during the purification, which step it is lost in

• Additionally, since different buffers containing varying salts are used through out the purification process we want to determine if anions can affect heat stability

Hypothesis• Based off last semester’s data, it appears there is a

protein in the cell lysate that increases the thermal stability of AP compared to the almost completely pure Sigma Aldrich AP

• It is likely that the loss of disulfide bonds during heating is what leads to the loss of activity, and since Dsb proteins can increase the stability of these bonds, we believe this is a possible protein that may be present in the cell lysate but not in the purified AP

• Additionally, we think that the presence of MgCl2 in the buffer used in the last step of purification is what leads to decreased activity after running a column and therefore this anion leads to decreased thermal stability of AP when compared to SO4

2-

Materials and Methods• Tested

– Pure Alkaline phosphatase obtained from Sigma Aldrich diluted in MgSO4 buffer and diluted in MgCl2 buffer

– Stage 1 enzyme from E. coli cell lysate– Stage 2 enzyme after heat denaturation– Stage 3 enzyme after dialysis and AmSO2

precipitation– Stage 4 enzyme after DEAE column

• Measured protein concentration using Bradford reagent and stock BSA for a standard curve

Materials and Methods cont.• Temperatures:

– Samples were heated for 30 min at 85, 90, and 95° C using heat blocks

• Times:– During the 30 minutes of heating, each sample’s

activity was measured at 5, 10, 15, 20, 25, and 30 minutes

– Activity was also measured 24 hours after heating (samples were left at room temperature during this time period)

Materials and Methods cont.• Activity

– After removal from the heat block samples were centrifuged for 10 seconds and 40 uL were removed

– This sample was added to a cuvette containing 250 uL of 1 mM PNPP and 750 uL of Tris buffer at pH 8.0

– Activity was measured at 400 nm using the Cary50 spectrometer

• Analysis– Activity at each time point was calculated using the

extinction coefficient 0.0175 uM-1cm-1 and divided by activity before heating to determine percent activity

Baseline levels of Pure AP activityIn MgSO4 buffer In MgCl2 buffer

Protein Concentration 0.1003 mg/mL 0.0878 mg/mL

Units of Activity 1,603 U/mL 835.1 U/mLSpecific Activity 15,982 U/mg 9,512 U/mg

Purification Level of Alkaline PhosphataseStage Volume

(mL) Units/mL Total Units

[Protein] (mg/mL)

Total Protein

(mg)

Specific Activity (U/mg)

% Yield Purification level

1 60 751.4 45085 0.5642 33.85 1332 ------ ------2 21 661 13882 0.2512 5.275 2633 30.79 1.983 3.2 3273 10472 1.055 3.033 3453 23.23 2.594 4 553.5 2214 0.0954 0.3816 5802 4.91 4.36

Effects of Salt on Heat Stability

0 5 10 15 20 25 300

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120

MgSO4-85MgCl2-85MgSO4-90MgCl2-90MgSO4-95

Time (min)

Perc

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Effects of Salt on Activity Recovery

0 5 10 15 20 25 300

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120 Pure AP in MgSO4 buffer

Time (min)

Perc

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0 5 10 15 20 25 300

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120Pure AP in MgCl2 buffer

85 degrees

85-After 24 hours

90 degrees

90-After 24 hours

95 degrees

95- After 24 hours

Time (min)

Perc

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Heat Stability During Purification Process

0 5 10 15 20 25 300

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140 85 degrees

Time (min)

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0 5 10 15 20 25 300

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120 90 degrees

Time (min)

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0 5 10 15 20 25 300

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120 95 degrees

SigmaStage 1Stage 2Stage 3Stage 4

Time (min)

Perc

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Ability to regain activity

0 5 10 15 20 25 300

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Stage 1

85 degrees85-After 24 hours90 degrees90-After 24 hours95 degrees95- After 24 hours

Time (min)

Perc

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Stage 2

85 degrees85-After 24 hours90 degrees90-After 24 hours95 degrees95- After 24 hours

Time (min)

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Ability to regain activity

0 5 10 15 20 25 300

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Pure AP in MgSO4 buffer

Time (min)

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Stage 3

Time (min)

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Stage 4

85 degrees85-After 24 hours90 degrees90-After 24 hours95 degrees

Time (min)

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Further Evidence

MarkerS 1 S 2 S 3 S 4

Marker

Alkaline Phosphatase

Conclusions• What anion is present in the buffer makes a

difference in Alkaline Phosphatase activity and its thermal stability. Chloride anions clearly lower the activity and ability to regain activity of the enzyme when compared to sulfate anions.

• This may be a concern during the purification process since different ions are used in the buffers at different steps.

• It is possible that sulfate anions are what lead to the thermal stability of alkaline phosphatase at 85° C

Conclusions cont.• We believe there is a protein present in the

cell lysate that is not present in Sigma Aldrich purified AP that contributes to the thermal stability of alkaline phosphatase

• It appears that this protein works optimally at around 90 °C but is less able to function by 95° C

• It is our conclusion that we lose some of this protein during the purification process but it is still present in the stage 4 enzyme

• It is possible that this protein is DsbC

Remaining Questions• How would the stage 4 enzyme’s thermal

stability and ability to regain activity be affected if we dialyze it with a MgSO4 buffer?

• If a SDS-Page gel was run for the stage 4 enzyme, silver stained, and the bands left excised and processed with mass spectroscopy would they be identified as DsbC or another protein?

• Alternatively, if a western blot was run and probed with a DsbC antibody would a band appear in the stage 4 lane?

References• Kurokawa, Yoichi, Hideki Yanagi, and Takashi Yura.

"Overexpression of Protein Disulfide Isomerase DsbC Stabilizes Multiple-Disulfide-Bonded Recombinant Protein Produced and Transported to the Periplasm in Escherichia Coli." Applied and Environmental Microbiology 66.9 (2000): 3960-965. PubMed. Web. 22 Apr. 2011.

• Poe, Richard W., Vani S. Sangadala, and John M. Brewer. "Effects of Various Salts on the Steady-state Enzymatic Activity of E. Coli Alkaline Phosphatase." Journal of Inorganic Biochemistry 50.3 (1993): 173-80. ScienceDirect. 12 Apr. 2001. Web. 22 Apr. 2011.