Profiling and quantitative evaluation of three nickel-coated magnetic matrices for purification of recombinant proteins: helpful hints for the optimized nanomagnetisable matrix preparation

Abstract

Background: Several materials are available in the market that work on the principle of protein magnetic fishing by their histidine (His) tags. Little information is available on their performance and it is often quoted that greatly improved purification of histidine-tagged proteins from crude extracts could be achieved. While some commercial magnetic matrices could be used successfully for purification of several His-tagged proteins, there are some which have been proved to operate just for a few extent of His-tagged proteins. Here, we address quantitative evaluation of three commercially available Nickel nanomagnetic beads for purification of two His-tagged proteins expressed in Escherichia coli and present helpful hints for optimized purification of such proteins and preparation of nanomagnetisable matrices.

Results: Marked differences in the performance of nanomagnetic matrices, principally on the basis of their specific binding capacity, recovery profile, the amount of imidazole needed for protein elution and the extent of target protein loss and purity were obtained. Based on the aforesaid criteria, one of these materials featured the best purification results (SiMAG/N-NTA/Nickel) for both proteins at the concentration of 4 mg/ml, while the other two (SiMAC-Nickel and SiMAG/CS-NTA/Nickel) did not work well with respect to specific binding capacity and recovery profile.

Conclusions: Taken together, functionality of different types of nanomagnetic matrices vary considerably. This variability may not only be dependent upon the structure and surface chemistry of the matrix which in turn determine the affinity of interaction, but, is also influenced to a lesser extent by the physical properties of the protein itself. Although the results of the present study may not be fully applied for all nanomagnetic matrices, but provide a framework which could be used to profiling and quantitative evaluation of other magnetisable matrices and also provide helpful hints for those researchers facing same challenge.

Figure 1

Densitometric analysis of recombinant protein expression. ProT (A) and Mre11 (B) recombinant proteins were expressed in E.coli and their relative expression in the soluble fraction of cell lysate were determined by densitometry using AlphaEase software.

Figure 2

SDS-PAGE analysis of flowthrough fractions of His-recombinant proteins bound onto the different concentrations of three Nickel-coated magnetic matrices. His-ProT and His-Mre11 recombinant proteins in soluble cell extract (SCE) of E.coli were bound to increasing concentrations of magnetic matrices, SiMAC-Nickel, SiMAG/N-NTA/Nickel and SiMAG/CS-NTA/Nickel, and flowthrough fraction of each matrix at each concentration was subjected to SDS-PAGE analysis. The target proteins are shown by black arrows.

Figure 3

Specific binding capacity of three Nickel-magnetic matrices for two His-tagged recombinant proteins. After binding of His-tagged recombinant proteins, His-ProT and His-Mre11, onto the Nickel-magnetic matrices, SiMAC-Nickel, SiMAG/N-NTA/Nickel and SiMAG/CS-NTA/Nickel, flowthrough fractions (FT) were subjected to SDS-PAGE analysis. Percent of band density in FT subtracted from 100% was defined as specific binding capacity. His-ProT (1), His-Mre11 (2), SiMAC-Nickel (A), SiMAG/N-NTA/Nickel (B) and SiMAG/CS-NTA/Nickel (C).

Figure 4

Comparison of purification yield and protein recovery of three Nickel-magnetic matrices for His-ProT and His-Mre11 recombinant proteins. Purification yield was defined as the sum of the percents of the specific band densities at four elution steps (E1-4). Recovery percent was calculated as the percent of purification yield divided by specific binding capacity. SiMAC-Nickel (A), SiMAG/N-NTA/Nickel (B) and SiMAG/CS-NTA/Nickel (C).

Figure 5

Effect of imidazole concentration on elution of recombinant proteins from three Nickel magnetic matrices. His-ProT and His-Mre11 recombinant proteins were bound onto the different concentrations of SiMAC-Nickel, SiMAG/N-NTA/Nickel and SiMAG/CS-NTA/Nickel magnetic matrices. Elution fractions collected by increasing concentrations of imidazole were subjected to SDS-PAGE analysis. RF: Residual fraction.

Figure 6

Percent loss of target recombinant proteins purified by three Nickel magnetic matrices. Percent of the recombinant proteins, His-ProT and His-Mre11, lost during purification process by SiMAC-Nickel, SiMAG/N-NTA/Nickel and SiMAG/CS-NTA/Nickel magnetic matrices was calculated as described in materials and methods. Comparison was made between three matrices for each protein. SiMAC-Nickel (A), SiMAG/N-NTA/Nickel (B) and SiMAG/CS-NTA/Nickel (C).

Figure 7

Western blot analysis of purified His-ProT and His-Mre11 recombinant proteins. Elution fractions of His-ProT and His-Mre11recombinant proteins purified by 4 mg/ml SiMAG/N-NTA/Nickel magnetic matrix were subjected to SDS-PAGE. Bands were transferred to nitrocellulose membrane and specific bands were detected by antibodies directed against 6His tag by ECL system. 1-3 indicated the fractions eluted by 0.05, 0.1 and 0.25 M imidazole, respectively.