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1Department of Life Sciences and National Institute for Biotechnology in the Negev, Ben-Gurion University
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MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamAΔ41) from M. magneticum AMB-1.
Zeytuni, N., Zarivach, R. Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamAΔ41. J. Vis. Exp. (37), e1844, doi:10.3791/1844 (2010).
1. Cloning and Expression of mamA Gene in E. coli
The mutant gene mamAΔ41 was amplified using the polymerase chain reaction (PCR) from genomic DNA of Magnetospirillum magneticum AMB-1, with primers: 5'-GCATTACGCATATGGACGACATCCGCCAGGTG-3' and 5'-GCGCGGCAGCCATA-TGGCATACG-3'. In the amplified DNA fragments, an NcoI site was introduced at the initiation codon ATG and the termination codon was replaced with a ScoI site. The fragments were digested with NcoI and SacI and cloned into the respective sites of pET52b(+), giving rise to pET52bMamAΔ41-AMB1. In this construct, the mamAΔ41 gene was fused in-frame with the 10-His tag at the C-terminus. The plasmid was electroporated into E. coli strain BL21. E. coli strain BL21 harboring pET52bMamAΔ41 was grown in auto-induction (15) medium containing ampicillin (50 mg/ml) 310°K for 3 hours. The cultivation temperature was then shifted from 310° to 300°K and maintained for an additional 48 h at 300°K. The cells were harvested by centrifugation at 5465g for 10 min at 277°K. 8 liters culture produced 60 grams of wet cell pellet.
2. Bioinformatics Calculations
Calculations of molecular weight (MW), according to amino acids sequence and the predicted absorption of 1mg/1ml of protein in 280nm, using the ProtParam server (http://www.expasy.ch/tools/protparam.html). For 10His-Tag-MamAΔ41 the MW is 22529 Da (240 amino acids) and for MamAΔ41 (with 9 amino acids left after His-tag removal by Thrombin) the Mw is 20596.5Da (187 amino acids). The predicted absorption of 1mg/1ml of protein in 280nm for 10His-Tag-MamAΔ41 is 0.595 and for MamAΔ41 is 0.579. In addition, the amino acid sequence does not contain any cysteine residues. Therefore, reducing agents are not required during the purification process.
3. Purification of MamAΔ41
4. Representative Results
When this protocol is done correctly one should get highly purified, size homogenous and concentrated protein samples. These protein samples are then ready for crystallization trials as well as biochemical studies, such as enzyme kinetics, binding affinity and more. Described here are representative results of main steps in the purification protocol. SDS-PAGE analysis of elution profile of Ni-NTA affinity column should reveal highly over expressed protein in the soluble fraction at appropriate MW of ~22kDa (Figure 1). This SDS-PAGE analysis should also reveal minimum if any protein in the unbounded proteins flow-through, wash flow-through and ultra-centrifugation pellet sample. If large bands of the appropriate protein do appear in these samples one should consider wrong buffer preparation, problems in cell disruption or problems in culture auto-induction conditions and growth.
Ion exchange chromatography is preformed in order to separate between our desirable protein to other E.coli proteins that were bound to the nickel resin (due to electrostatic interactions or histidine/negative amino acids rich protein loops) and uncut His-Tagged desirable protein. This column separates proteins according to their binding affinity to the positively charged resin under increasing NaCl concentrations. Highly negatively charged protein will elute in higher NaCl concentrations appose to moderate negatively charged ones. The advantages of this column are high flow rate and binding capacity. The ion exchange chromatogram (Figure 2) reveals a good separation between 3 proteins populations in increasing NaCl concentration. SDS-PAGE analysis is needed in order to determine MW of each population, isolating the desirable one and evaluation whether further purification steps are needed. The first population is MamAΔ41 (~20 kDa) while the second population is MamAΔ41+His Tag (~22kDa) that was not cleaved by bovine thrombin and the third population is undetectable in SDS-PAGE due to low concentration. If the proteins peaks are not separated clearly one should consider wrong buffer preparation or changing the slope of NaCl gradient.
Size exclusion chromatography is preformed in order to separate between our desirable protein to other E.coli cell proteins that were bound to the Ni-NTA resin and were not separated during ion exchange chromatography. This column separates proteins according to their size. Large proteins will elute in smaller elution volumes opposed to small ones which will be eluted in larger volumes. The column chromatogram (Figure 3) reveals a good separation of the desirable protein as one main population. The population appears to elute in appropriate monomer size with a MW of ~20 kDa. The presence of ~80 kDa band (E.coli typical protein that binds Ni-NTA resin) is detected in SDS-PAGE prior to column loading and disappears during this run. Since the concentration of this ~80 kDa protein is low to begin with, its population does not appear in the chromatogram due to its dilution and SDS-PAGE analysis is needed to determine the purification efficiency. We recommend loading a diluted and concentrated sample of the main peak to the SDS-PAGE so one could surely determine the presence of a pure protein in the appropriate MW. By this stage the protein is purified and its homogeneity should be evaluated by MALDI-TOF. This analysis revealed a protein in MW of 20347 Da and another in Mw of 10176 Da (Figure 4). The ~10 kDa protein is the ~20 kDa protein doubled charged. The predicted MW of MamAΔ41 with the 9 amino acids left after His-Tag removal was 20596.5 Da. By comparing it to the obtained MALDI-TOF MW we found that they are 248 Da apart. This dissimilarity can result from MALDI-TOF measurement errors and/or due to common degradation of the first methionine and the second glycine. To conclude, the protein is highly purified and can be used for further experiments.
Table 1. Buffer formulations.
Figure 1. Representative SDS-PAGE analysis of Ni-NTA column purification. From right to left; P- ultra-centrifuge pellet (3.11 protocol step), W- wash (3.12 protocol step), U- unbounded proteins (3.10 protocol step), E-five lanes of elution samples (3.14 protocol step), M - protein marker (numbers indicate MW). Arrow indicates MamAΔ41.
Figure 2. Analysis of ion exchange chromatography step (a) Ion exchange (MonoQ column) chromatogram; Blue - absorption 280nm, represents protein concentration, Green- NaCl concentration, Red - indication of fractions collected. (b) Representative SDS-PAGE analysis of the ion exchange purification step. From left to right; M - protein marker (numbers indicate Mw), A6 - first peak fraction, A8- second peak fraction.
Figure 3. Size exclusion chromatography analysis (a) Size exclusion (Superdex 200 column) chromatogram; Blue - absorption 280nm, represents protein concentration, Red indication of fractions collected. (b) Representative SDS-PAGE analysis of size exclusion purification step. From left to right; M- protein marker (numbers indicate MW), PreI- pre-injected sample, A4-6 combined fraction collected from the first peak, A4-6D- Diluted fraction collected from the peak.
Figure 4. Matrix-assisted laser desorption/ionization (MALDI-TOF) mass spectrum of purified MamAΔ41. The matrix is Sinapic acid (SA). MamAΔ41 shown at 20347 Da, also shown is the doubled charged species of MamAΔ41 at 10176 Da.
Protein purification is the main step in any proteins biochemical or structural studies. Since each protein is unique with its own behavior, one needs to define its properties and modify its purification accordingly. Protein target should be analyzed as a first step toward purification using bioinformatics tools. They are used to calculate the target iso-electric point, assess its need for reducing/oxidizing environment and its need for special ions/ligands. There are several critical modifications of our protocol which reflect the target uniqueness. These modifications include buffer, working temperatures and column adjustments.
The first critical modification is the buffer in use (section 3.3) such buffers need to be altered to reflect the correct pH range, ions/ligands and ionic strength needed for protein stability and functions. If the target shows any sign of instability (degradation, precipitation or loss of function) one need to test and search for other more suitable buffers. Another critical issue is the purification speed and working temperatures. As a mean to avoid protein degradation (due to proteases or protein inner instability) rapid purification should performed at 277°K (including the use of cold buffers, rotors and columns).
Affinity purification steps as well as other chromatography stages can be perform on various resin sources. Affinity resin can be protein loaded using several techniques and it should be altered based on the local settings. If the resin is loaded by passing the protein solution over it, one should use slow flow (1-1.5 ml/min) to ensure maximum protein capture. For batch binding (mixing the resin with the protein solution) one need to allow ~10 min incubation at room temperature and longer (up to 1 hr) at 277°K. Based on the affinity strength, resin wash could be done under various imidazole concentrations.
Overall, alteration of each step in this protocol should take place in order to purify efficiently the unique protein target taking under consideration this purification scheme as a guide.
We acknowledge Dr. Amir Aharoni for his support and Geula Davidov, Noam Grimberg and Chen Guttman for their advice and comments.
|French Press||Equipment||Thermo Fisher Scientific, Inc.||FA-078A|
|Pressure cell||Equipment||Thermo Fisher Scientific, Inc.||FA-032|
|Ultra-centrifuge||Equipment||Sorvall, Thermo Scientific||Discovery 90SE|
|Rottor||Equipment||Beckman Coulter Inc.||Ti60|
|Ultra-centrifuge tubes; PC-Bottle+Cap Assay 26.3ml||Equipment||Beckman Coulter Inc.||BC-355618|
|2.5cm diameter, Glass Econo-Column Chromatography Columns||Equipment||Bio-Rad||737-2521|
|Ni-NTA His Bind resin||Equipment||Novagen, EMD Millipore||M0063428|
|Spectrophotometer||Equipment||Amersham||Ultraspec 2100 pro|
|Fast Performance Liquid Chromatography- AKTA purifier 10||Equipment||GE Healthcare||28-4062-64|
|Ion exchange column – MonoQ 4.6/100 PE||Equipment||GE Healthcare||10025543|
|Size exclusion pre-packed column-HiLoad 26/60 Superdex 200||Equipment||GE Healthcare||17-1071-01|
|Centricon - Vivaspin15 – 10,000 MWCO||Equipment||Sartorius AG||VS1501|
|Table centrifuge||Equipment||Thermo Fisher Scientific, Inc.||IEC CL30R|
|MALDI-TOF||Equipment||Bruker Corporation||Reflex IV|
|Tris-HCl (hydrotymethyl) aminomethane||Reagent||BioLab||20092391|
|EDTA free protease inhibitors cocktail||Reagent||Sigma-Aldrich||P-8849|
|Dnase I (Deoxyribonuclease I)||Reagent||Sigma-Aldrich||DN-25|
|Bovine Thrombin||Reagent||Fisher Scientific||BP25432|
|Soudim Dodecyl Sulfate (SDS)||Reagent||BioLab||19822391|
|PageRuler Prestained Protein Ladder||Reagent||Fermentas||SM0671|
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