B. Sample preparation
3.7 Production and Purification of MBP fusion proteins
3.7.2 Purification of MBP Fusion Proteins by Affinity Chromatography
MBP-LcaA MBP-preLcaA
Figure 3.18. SDS-PAGE analysis of the amylose affinity chromatography used for the purification of MBP-LcaA and MBP-preLcaA. Lane 1: BIORAD Precision Plus Molecular Weight Marker (BioRad, South Africa), lane 2: crude extract of clone pLcaA containing the MBP-LcaA fusion protein, lane 3: flow through, lane 4: wash, lane 5: empty, lane 6: crude extract of clone pPreLcaA containing the MBP-preLcaA fusion protein, lane 7: flow through, lane 8: wash. Bands representing fusion proteins are highlighted in the respective lanes. Dark bands in lanes 2 and 6 indicate that the concentration of MBP-LcaA and MBP-preLeuA is high. These bands are also present in the flow through and wash samples indicating poor binding of each fusion protein to the amylose resin.
Following binding of MBP-LcaA and MBP-preLcaA onto the amylose matrix, the fusion proteins were eluted under mild conditions using maltose. Maltose has an affinity to bind to amylose and thereby displaces the bound MBP-LcaA and MBP-preLcaA by competitive binding (Hage, 1999). In general practice, MBP fusion proteins are usually eluted within the first five fractions (Lauritzen et al., 1991). For the elution of MBP-LcaA and MBP-preLcaA nine 3 ml fractions were collected for each fusion protein. These fractions recovered during purification were analyzed by 10 % SDS-PAGE in order to determine if the procedure was successful in purifying both fusion proteins. The expected results for SDS-PAGE analysis is a single band representing each fusion protein. The results obtained for fractions collected for MBP-LcaA and MBP-preLcaA are shown in figures 3.19(a) and 3.20(a) respectively. The first three fractions of MBP-LcaA and four fractions of MBP-preLcaA collected, resulted in the elution of many non-specific proteins for both purification procedures. This can be due to low stringency washes of the column. Furthermore, the majority of the fusion proteins were eluted within these fractions and this is seen by the intense bands produced in lane 3 and 4 on figures 3.19(a) and 3.20(a). These fractions required further purification before subsequent analysis and this can be achieved by passing fractions 3 and 4 of both fusion proteins through the amylose column again, washing the column, and eluting new fractions. Pure MBP-LeuA was eluted from the fourth fraction collected and this seen by the presence of single bands (~
46.433 kDa) in lanes 5 to 10 on figure 3.20(a). MBP-preLcaA was eluted from fraction 5 to 9.
Single bands of ~ 49.088 kDa are seen in lanes 6 to 10 on figure 3.20(a). The intensity of single bands for MBP-LcaA and MBP-preLcaA is high, indicating that fusion proteins are concentrated within these fractions. Therefore, subsequent concentration steps by freeze drying were not required.
The overall purification procedure for each fusion protein was monitored at 280 nm. This involved taking absorbance readings of aliquots of each crude extract, flow through, washes, and the fractions collected. Chromatograms for the affinity separation of MBP-LcaA and MBP-preLcaA were then constructed using these values. These are represented in figures 3.19(b) for the purification of MBP-LcaA, and figure 3.20(b) for MBP-preLcaA. From these chromatograms, it is seen that the protein concentration of each fusion sample is decreasing at the flow through and wash steps. This is due to non-specific protein passing through the amylose column. The A280 values then begin to peak at particular fractions, which indicates those fractions that contain the highest concentration of each MBP fusion protein. However, analysis of the chromatograms together with the SDS-PAGE containing the fractions
collected, indicate that peaks at fractions 1 and 2 for MBP-LcaA, and fractions 2, 3, and 4 for MBP-preLcaA, are due to non-specific proteins. This is due to the multiple bands seen in figure 3.19(a) (lanes 3 and 4), and figure 3.20(a) (lanes 3, 4, and 5). Peaks for fraction 6 and 7 for MBP-LcaA and MBP-preLcaA and single bands for these fractions (figures 3.20a and 3.20a) represent those fractions that contain the highest concentration of the respective fusion proteins. From the results seen in figures 3.19 and 3.20, it can be concluded that affinity chromatography was successful in isolating pure forms of each MBP fusion protein.
(a) (b)
Figure 3.19. (a) SDS-PAGE analysis of MBP-LcaA fractions collected by amylose affinity chromatography. Lane 1: BIORAD Precision Plus Molecular Weight Marker (BioRad, South Africa) , lane 2 to lane 10: fraction 1 to 9 of MBP-LcaA. Fractions containing pure MBP-LeuA were obtained from fraction 4 onwards; (b) A chromatogram constructed for the affinity purification of MBP-LcaA. The absorbance at 280 nm of each component of the purification i.e. crude extract, flow through, washes, and fractions 1 to 9 were determined. Fractions 6 and 7 which are indicated by an arrow contain the concentration of MBP-LcaA.
MBP-LcaA ~ 46.433 kDa
Highest concentrations of MBP-LcaA
(a) (b)
Figure 3.20. (a) SDS-PAGE analysis of MBP-preLcaA fractions collected by amylose affinity chromatography. Lane 1: BIORAD Precision Plus Molecular Weight Marker (BioRad, South Africa), lane 2 to lane 10: fraction 1–9 of MBP-preLcaA. Pure fractions of MBP-preLcaA were obtained from fraction 5 onwards; (b) A chromatogram constructed for the affinity purification of MBP-preLcaA. The absorbance at 280 nm of each component of the purification i.e. crude extract, flow through, washes, and fractions 1 to 9 were determined. Fractions 6 and 7 which are indicated by an arrow contain the concentration of MBP-preLcaA.
MBP-preLcaA ~ 49.088 kDa
Highest concentrations of MBP-preLcaA