Team:Hong Kong HKU/BMCcargocopurification

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Latest revision as of 02:40, 18 October 2014

BMC-cargo co-purification

BMC-cargo co-purification

With pETES-mCherry in BL21(DE3), we then used 0.2mM IPTG to induce the expression of proteins. The successful expression and proper functioning of mCherry protein is readily apparent: the culture was cherry-red in color after overnight induction. (Fig. 6)

We then expanded the scale to 1.5L of cell culture in LB, and carried out the protein extraction procedure as previously described. Clarified, filtered soluble protein fraction was purified as described previously using a HiTrap Chelating column, but this time on a 5ml scale. (Fig. 10), indicating significant loss.) Elution was then carried out with 30%, 50%, 100% gradient Elution buffer and stripped as described previously, with all fractions collected for analysis. Whenever necessary, 100% glycerol was added to a final concentration of 10% and stored at -70°C overnight, and thawing was done slowly on ice at 4°C.

Fig. 10. Sample that flowed through the Ni-Affinity column shows significant amount of mCherry, which is lost.

Gel filtration was done using Superdex 10/300 GL 200 from GE HealthSciences, controlled and monitored by an AKTApurifier system. Prior to gel filtration, the column was washed once with MiliQ water and then equilibrated with the 30% elution buffer, and the sample was centrifuged at max speed using a tabletop centrifuge for 10min at 4°C to remove any precipitate. 200μl of sample, which we chose the 30% eluent as it is the only one which shows the red color of mCherry, was loaded onto the column, and fractions at 0.5ml intervals were collected (Fig. 8).

Four peaks of the UV Abs₂₈₀ were revealed. Fractions containing the peak regions were analyzed on an SDS-PAGE. However, due to time constraints, the gel pictures was not ready for posting on the Wiki so do stay tuned.

Two gel filtration experiments has been carried out, and they showed similar peak patterns (Fig. 11).

Standards (Blue dextran 2,000, Ferritin, Aldolase, Ovalbumin, Albumin, Chymotrypsinogen A, Ribonuclease A) with known molecular weights were loaded on to the column under the same conditions and buffer composition, the elution volume of Blue dextran 2,000 was defined to be the void volume (Vo), and each of the standards’ distribution coefficient (Kav) was calculated and used to plot a calibration curve.(Fig. 12).

Item	Elution volume	Ve-Vo(ml)	Molecular weight(Da)	Kav	In(MW)
Blue Dextran 200	8.01	0	2000000	0	14.50865774
Ferritin	10.52	2.51	440000	0.156973108	12.99453001
Aldolase	13.25	5.24	158000	0.327704816	11.97035031
Albumin	14.16	6.15	67000	0.384615385	11.1124479
Ovalbumin	15.28	7.27	43000	0.454659162	10.66895539
Chymotrypinogen A	16.99	8.98	25656	0.561601001	10.15253274
Ribonuclease A	17.81	9.8	13700	0.612883052	9.525151112

The area-under-curves of the 4 peaks of the 1st gel filtration attempt were integrated and their corresponding elution volumes (Ve) determined. By applying the equation obtained from the calibration curves, their molecular weights were determined to be:

	Ve	Ve-Vo	Kav - (Ve-Vo)/(Vt-Vo)	Kav applied to calibration curve equation	Observed molecular weight (in kDa):
Peak 1	7.72	-0.29	-0.02	14.36	1715.541273
Peak 2	10.52	2.51	0.16	13.02	450.6858856
Peak 3	12.76	4.75	0.30	11.95	154.6864192
Peak 4	15.29	7.28	0.46	10.74	46.22792313

where Vt is the total volume of the volume.

Remarkably, the peak 1 has a void volume even less than that of Blue dextran 2,000, which indicates its molecular weight exceeds 2,000kDa, leading us to speculate that it could either be the BMC itself, or a very non-specific, huge protein aggregate. The proteinaceous nature of the first peak eluent was confirmed using a Bradford assay. The obvious next steps are: (1) SDS-PAGE analysis of the collected fractions corresponding to the peaks, which is in progress, and (2) re-run a gel filtration analysis after denaturation with Superdex-compatible denaturants, such as 6M Urea of 8M Guanidium chloride. Controls with BL21(DE3) carrying an empty pET28a could also be done in parallel for comparison. These data, whether or not the purification of the intact BMC is successful or not, can certainly reveal important information on both the chemical and physical properties of the BMCs. Furthermore, as we have seen from previous results that there are other bands that appears in the Ni affinity column-purified BMCs, such experiments can reveal and separate novel proteins that associates with BMCs, which is invaluable for understanding more about the BMC’s interactions with the host organism’s proteins and other components.

@@ Line 22: / Line 22: @@
-BMC-cargo co-purification
+<b>BMC-cargo co-purification</b>
 <br><br>
-With pETES-mCherry in BL21(DE3), we then used 0.2mM IPTG to induce the expression of proteins. The successful expression and proper functioning of mCherry protein is readily apparent: the culture was cherry-red in color after overnight induction. (Fig. 6)
+With pETES-mCherry in BL21(DE3), we then used 0.2mM IPTG to induce the expression of proteins. The successful expression and proper functioning of mCherry protein is readily apparent: the culture was cherry-red in color after overnight induction. <font color="red">(Fig. 6) </font>
 <br>
 <img src="https://static.igem.org/mediawiki/2014/9/9b/Fig._6.JPG" width="800px">
 <br>
-We then expanded the scale to 1.5L of cell culture in LB, and carried out the protein extraction procedure as previously described. Clarified, filtered soluble protein fraction was purified as described previously using a HiTrap Chelating column, but this time on a 5ml scale. (Fig. 7)
+We then expanded the scale to 1.5L of cell culture in LB, and carried out the protein extraction procedure as previously described. Clarified, filtered soluble protein fraction was purified as described previously using a HiTrap Chelating column, but this time on a 5ml scale. <font color="red>(Fig. 7)</font>
 <br>
 <img src="https://static.igem.org/mediawiki/2014/9/9c/Fig._7.JPG" width="800px>
@@ Line 38: / Line 38: @@
 The induction was carried out as previously described: the BL21(DE3) were grown at 37°C 200rpm for 4 hours until OD ~0.8-1 in LB supplemented with 50mg/ml Kanamycin, and 1ml of the pre-inoculant was separately added into three 500ml LB supplemented with Kanamycin in 2L flasks to ensure proper aeration for mCherry protein activation, and allowed to grow to OD ~0.8, and IPTG was subsequently added to a final concentration of 0.2mM and induction was carried out overnight at room temperature, 200rpm.
 <br><br>
-The cells were pelleted, collected, washed, and sonication buffer added. Sonication was carried out with the details as described previously. The sample was clarified two times by centrifugation and filtered. The total soluble proteins were loaded onto a prepared 5ml HiTrap Chelating column, both flow-through and wash-through were collected for analysis (as they shows intense red color of mCherry (Fig. 10), indicating significant loss.)  Elution was then carried out with 30%, 50%, 100% gradient Elution buffer and stripped as described previously, with all fractions collected for analysis. Whenever necessary, 100% glycerol was added to a final concentration of 10% and stored at -70°C overnight, and thawing was done slowly on ice at 4°C.
+The cells were pelleted, collected, washed, and sonication buffer added. Sonication was carried out with the details as described previously. The sample was clarified two times by centrifugation and filtered. The total soluble proteins were loaded onto a prepared 5ml HiTrap Chelating column, both flow-through and wash-through were collected for analysis (as they shows intense red color of mCherry <font color="red">(Fig. 10)</font>, indicating significant loss.)  Elution was then carried out with 30%, 50%, 100% gradient Elution buffer and stripped as described previously, with all fractions collected for analysis. Whenever necessary, 100% glycerol was added to a final concentration of 10% and stored at -70°C overnight, and thawing was done slowly on ice at 4°C.
 <br>
 <img src="https://static.igem.org/mediawiki/2014/1/1d/Fig._10.JPG" width="800px">
@@ Line 44: / Line 44: @@
 Fig. 10. Sample that flowed through the Ni-Affinity column shows significant amount of mCherry, which is lost.
 <br><br>
-Gel filtration was done using Superdex 10/300 GL 200 from GE HealthSciences, controlled and monitored by an AKTApurifier system. Prior to gel filtration, the column was washed once with MiliQ water and then equilibrated with the 30% elution buffer, and the sample was centrifuged at max speed using a tabletop centrifuge for 10min at 4°C to remove any precipitate. 200μl of sample, which we chose the 30% eluent as it is the only one which shows the red color of mCherry, was loaded onto the column, and fractions at 0.5ml intervals were collected (Fig. 8).
+Gel filtration was done using Superdex 10/300 GL 200 from GE HealthSciences, controlled and monitored by an AKTApurifier system. Prior to gel filtration, the column was washed once with MiliQ water and then equilibrated with the 30% elution buffer, and the sample was centrifuged at max speed using a tabletop centrifuge for 10min at 4°C to remove any precipitate. 200μl of sample, which we chose the 30% eluent as it is the only one which shows the red color of mCherry, was loaded onto the column, and fractions at 0.5ml intervals were collected <font color="red">(Fig. 8)</font>.
 <br>
 <img src="https://static.igem.org/mediawiki/2014/7/72/Fig._8.jpg" width="800px">
@@ Line 50: / Line 50: @@
 Four peaks of the UV Abs<sub>280</sub> were revealed. Fractions containing the peak regions were analyzed on an SDS-PAGE. However, due to time constraints, the gel pictures was not ready for posting on the Wiki so do stay tuned.
 <br><br>
-Two gel filtration experiments has been carried out, and they showed similar peak patterns (Fig. 11).
+Two gel filtration experiments has been carried out, and they showed similar peak patterns <font color="red">(Fig. 11)</font>.
 <br>
 <img src="https://static.igem.org/mediawiki/2014/9/93/Fig._5.png" width="800px">
 <br>
-Standards (Blue dextran 2,000, Ferritin, Aldolase, Ovalbumin, Albumin, Chymotrypsinogen A, Ribonuclease A) with known molecular weights were loaded on to the column under the same conditions and buffer composition, the elution volume of Blue dextran 2,000 was defined to be the void volume (Vo), and each of the standards’ distribution coefficient (Kav) was calculated and used to plot a calibration curve.(Fig. 12).
+Standards (Blue dextran 2,000, Ferritin, Aldolase, Ovalbumin, Albumin, Chymotrypsinogen A, Ribonuclease A) with known molecular weights were loaded on to the column under the same conditions and buffer composition, the elution volume of Blue dextran 2,000 was defined to be the void volume (Vo), and each of the standards’ distribution coefficient (Kav) was calculated and used to plot a calibration curve.<font color="red">(Fig. 12)</font>.
 <br>
 <table style="width:100%">
@@ Line 99: / Line 99: @@
    <tr>
      <td>Ovalbumin</td>
-     <td>15.28/td>
+     <td>15.28</td>
      <td>7.27</td>
      <td>43000</td>
@@ Line 123: / Line 123: @@
 </table>
 <img src="https://static.igem.org/mediawiki/2014/5/51/Fig._12.png" width="800px">
-<br>
+<br><br>
 The area-under-curves of the 4 peaks of the 1st gel filtration attempt were integrated and their corresponding elution volumes (Ve) determined. By applying the equation obtained from the calibration curves, their molecular weights were determined to be:
 <br>