His-tag Protein Purification Kit

Instruction

This protocol is provided for the purification of His-tagged recombinant protein from the E. coli expression system under native conditions. For protein purification from other expression systems, except for different protein expression methods, the purification procedures with this product are similar. For first-time users, please follow the protocol strictly. A Simplified Purification Protocol is also provided for users who are familiar with the Nickel resin purification system.
1.Induced expression of His-tagged recombinant protein in E. coli
IPTG is most commonly used to induce protein expression in E. coli, as described below. Please refer to appropriate instructions for other inducing systems.
a. Transform His-tagged protein expression plasmid into E. coli competent cells designed for protein expression specifically. After overnight incubation, culture a single colony in 3ml or 10-20ml of LB medium containing appropriate antibiotics.
b. Add the overnight bacteria culture solution into the new LB medium preheated to 37℃ at a ratio of 1:20 (e.g. Add 5ml bacteria culture into 100ml of LB medium containing appropriate antibiotics).
Note 1: Scale up or down the LB medium, depending on your experimental requirements. For optimization of protein expression conditions, 3-10ml LB medium is generally used. For protein purification, 100ml or more should be considered.
Note 2: To get a better expression effect, we recommend a ratio of 1:100, but it will take longer for the culture to reach the optimal OD600 required for IPTG induction.
c. Cultivate E. coli at 37℃ with vigorous shaking in a shaker until the OD600 of bacteria culture reaches 0.6.
d. Add IPTG to bacteria culture at a final concentration of 1mM, and continue cultivation for 4-5 hours with vigorous shaking at 37℃.
Note 1: The optimal IPTG concentration, duration of IPTG induction, and temperature during the induction should be determined experimentally for different proteins.
Note 2: A small amount of bacterial culture solution can be taken before and after IPTG induction to examine the induced expression level of His-tagged protein.
e. Harvest bacteria by centrifugation at 4000g for 20 minutes at 4℃, or 15,000g for 1 minute. Discard supernatant. Proceed to bacteria lysis immediately, or freeze the pellet at -20℃ or -80℃ for later use. The frozen bacteria should be thawed on ice for 15 minutes prior to bacteria lysis.
2.Small-scale purification of His-tagged protein under native conditions
This method is generally used for small-scale protein purifications or optimizing conditions for protein expression and purification on a large scale.
a. Following step 1e, add 100μl of lysis buffer to the bacteria pellet obtained per ml of LB culture. Resuspend the pellet completely by pipetting or vortex gently (avoid bubbles).
Note: Based on the expression level of His-tagged recombinant proteins, the volume ratio of bacteria culture to lysis buffer can be optimized between 25:1 and 5:1. When the expression level is very high, use 200μl of lysis buffer for bacteria pellet per ml of LB culture, while when expression level is very low, use 40μl of lysis buffer. After lysis, the lysate can be used directly for SDS-PAGE analysis. When necessary, an appropriate protease inhibitor can be added to the lysis buffer to minimize protein degradation.
b. Add lysozyme to bacteria suspension at a final concentration of 1mg/ml, mix gently, and incubate on ice for 30 minutes.
Note: A lysozyme stock of 100mg/ml prepared with lysis buffer can be used. Aliquot the lysozyme stock solution and store at -20℃.
c. Gently vortex several times during the incubation to ensure sufficient lysis of bacteria, avoiding bubble production.
d. Centrifuge at 15000g for 10 minutes at 4℃, take 10μl of supernatant for SDS-PAGE analysis, and collect the rest into another new centrifuge tube for protein purification.
e. Add 20μl BeyoGold™ His-tag Purification Resin (resuspend evenly prior to use) into the supernatant of lysate, and incubate for 30 minutes at 4℃ with agitation on a shaker to ensure a sufficient binding between His-tagged proteins and the resin.
Note: An incubation time of 30 minutes is usually sufficient for protein binding, but can be prolonged or even overnight under certain circumstances. Using the BeyoGold™ His-tag Purification Resin directly without pre-washing has been tested to work efficiently. However, protein yield can be increased by 5-20% if the resin is equilibrated 2-3 times with a bed volume of lysis buffer prior to use.
f. Pellet the resin by centrifugation at 1000g for 10 seconds at 4℃, take 20μl of supernatant for SDS-PAGE analysis and discard the rest.
g. Resuspend resin with 40μl of wash buffer by pipetting gently, and centrifuge at 1000g for 10 seconds at 4℃. Take 20μl of supernatant for SDS-PAGE analysis and discard the rest.
h. Repeat step g once.
i. Add 20μl of elution buffer and resuspend the resin by pipetting gently. Centrifuge at 1000g for 10 seconds at 4℃, and then collect the supernatant that contains the purified His-tagged protein.
j. To increase protein recovery, step i can be repeated twice and a total 60μl of purified protein can be obtained.
3.Large-scale purification of His-tagged proteins under native conditions
a. Following step 1e, add 4ml (2-5ml generally) of lysis buffer per gram of bacteria pellet to resuspend bacteria completely. If necessary, an appropriate amount of protease inhibitor can be added to the lysis buffer to minimize protein degradation.
b. Add lysozyme to bacteria suspension at a final concentration of 1mg/ml, mix gently, and incubate on ice for 30 minutes.
Note: A lysozyme stock of 100mg/ml prepared with lysis buffer can be used. Aliquot the lysozyme stock solution and store at -20℃.
c. Using a sonicator with a power of 200-300W, sonicate the solution on ice using six 10-second bursts at high intensity with a 10-second cooling period between each burst to keep the sample cold.
Note: The number of pulses, pulse duration, and pulse intensity should be optimized for each sample type according to the specific type of ultrasonic instrument.
d. (Optional) If the lysate is very viscous after sonication, add RNase A to 10 μg/ml and DNase I to 5 μg/ml, and incubate on ice for 10-15 minutes. Alternatively, draw the lysate through a syringe needle several times to break the viscous genomic DNA.
e. Centrifuge the lysate at 10,000g for 20-30min at 4℃. Transfer the supernatant to a new tube and keep it on ice. Take 20μl of supernatant for SDS-PAGE analysis.
Note: The supernatant must be completely clear before loading to BeyoGold™ His-tag Purification Resin. The presence of insoluble particulates will severely affect the purity of His-tagged proteins obtained subsequently. It is good practice to filter the lysates just prior to loading using a 0.45μm filter even if they have been filtered several days before the purification.
f. Take 1ml of BeyoGold™ His-tag Purification Resin (resuspend evenly prior to use), centrifuge at 1000g for 10 seconds at 4℃, and discard the supernatant. Add 0.5ml of lysis buffer to resuspend the resin evenly, centrifuge again, and discard the supernatant. Repeat resin equilibration 1-2 times with lysis buffer, centrifuge, and discard the supernatant.
Note: BeyoGold™ His-tag Purification Resin can also be used directly without equilibration, but protein yield may decrease by 5-20%.
g. Add 4ml of bacteria lysate obtained in step 2e to the equilibrated His-tag Purification Resin (about 0.5ml). Mix well and incubate for 60 minutes with gentle agitation on a side or horizontal shaker at 4℃.
h. Load the mixture of lysate and resin into a suitable empty chromatography column, such as Beyotime Affinity Chromatography Empty Column.
Note: Resin can be loaded into an Empty Affinity Chromatography Column either before or after protein binding. Loading resin after protein binding can ensure a sufficient binding of His-tagged protein with the resin, especially when the His-tag is partially blocked by the fusion protein or the expression of His-tagged recombinant protein is very low. If performing resin packing before protein binding, equilibrate the column with 0.5ml of lysis buffer 2-3 times after packing, and then load 4ml of lysate onto the column. The flow through of the lysate can be reloaded onto column 3-5 times to ensure sufficient binding of His-tagged proteins.
i. Remove the snap-off cap at the bottom of the column, and allow the liquid to flow out of the column by gravity. Collect 20 μl of flow-through liquid for SDS-PAGE analysis.
j. Wash the resin by adding 0.5-1ml of wash buffer and allow the liquid to flow out by gravity. Collect 20 μl of flow-through liquid for SDS-PAGE analysis. Repeat washing 5 times. Protein content in the flow-through can be examined by the Bradford method (Beyotime, P0006) to determine whether the washing is sufficient.
Note: Increase the number of washes 2-3 times to improve protein purity.
k. Elute protein 6-10 times, with 0.5ml of elution buffer each. Collect each eluate into different centrifuge tubes and determine the protein concentration of each eluate using the Bradford method. Pool the eluates containing the purified protein. Please refer to Figure 1 for the SDS-PAGE analysis of collected samples during the purification process.

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