For His-tagged protein purification using liquid chromatography systems
|Bead size||60–160 µm|
|Number of preps per run||1–24 samples per run|
|Special feature||Batch and column purification|
|Gravity flow or spin column||Gravity flow or automated|
|Binding capacity||Up to 50 mg/ml|
|Start material||Cell lysate|
|Yield||Depends on binding capacity|
This kit uses specially designed primers to amplify coding DNA sequence and supplement it with regulatory elements required for optimal transcription and translation in cell-free expression systems. In addition, specially designed 5' untranslated regions (UTRs) on the sense adapter primer sequences reduce the formation of secondary structure in the translation initiation region, one of the commonest causes of low expression rates. A His-or Strep-tag II can be added to either terminus, greatly simplifying protein purification and detection after expression.
The buffers of the Ni-NTA Fast Start Kit are based on recipes for the respective buffers for purification of 6xHis-tagged proteins under native or denaturing conditions listed in the QIAexpressionist handbook. Specific components have been added for optimized performance. The exact composition of the buffers in the Ni-NTA Fast Start Kit is confidential. However, the buffers listed in the Appendix Section of the QIAexpressionist are compatible with the Ni-NTA Fast Start Kit, and can also be used.
|The interaction of the 6xHis tag with Ni-NTA matrices is conformation independent||One-step purification can be carried out under native or denaturing conditions|
|Mild elution conditions can be used||Binding, washing, and elution are highly reproducible, and have no effect on protein structure. Pure protein products are ready for direct use in downstream applications|
|The 6xHis tag is much smaller than other commonly used tags||6xHis tags can be used in any expression system. The Tag does not interfere with the structure and function of the recombinant protein|
|The 6xHis tag is uncharged at physiological pH||The 6xHis tag does not interfere with secretion|
|The 6xHis tag is poorly immunogenic||The recombinant protein can be used without prior removal of the tag as an antigen to generate antibodies against the protein of interest|
|Using Factor Xa Protease, 6xHis tag can be easily and efficiently removed||The detagged protein can be used for crystallographical or NMR studies where removal of the 6xHis tag may be preferred|
|Some QIAexpress vectors feature a 6xHis-dihydrofolate reductase tag (6xHis-DHFR tag)||Small peptides fused to the 6xHis DHFR tag are stabilized while being expressed. The 6xHis-DHFR tag is not highly immunogenic in mouse and rat, so that peptides fused to the tag can be used directly for immunizations or epitope mapping|
The binding capacity of both resins is the same: up to 50mg/ ml mg 6xHis-tagged protein per ml of resin (2500 nmol @ ~20 kDa). The difference between them is the bead support, which determines pressure resistance and flow rate:
You can find a detailed comparison table in the Appendix at the back of the QIAexpressionist Handbook under the title 'Ni-NTA Matrices'.
The reuse of Ni-NTA Agarose and Ni-NTA Superflow resins depends on the nature of the sample and should only be performed with identical recombinant proteins. We recommend a maximum of 5 runs per column. After use the resin should be washed for 30 minutes with 0.5 M NaOH. Ni-NTA matrices should be stored in 30% ethanol to inhibit microbial growth.
If the Ni-NTA matrix changes from light blue to brownish-gray, the regeneration procedure described in the Appendix of the QIAexpressionist Handbook in section 'Reuse of Ni-NTA Resin' is recommended.
Compatibility of reagents with Ni-NTA matrices
|Tris, HEPES, MOPS||Buffers with secondary or tertiary amines will reduce nickel ions||
Up to 100 mM has been used successfully in some cases
Sodium phosphate or phosphate-citrate buffer is recommended
|EDTA, EGTA||Strip nickel ions from resin||Up to 1 mM has been used successfully in some cases, but care must be taken|
|beta-mercaptoethanol||Prevents disulfide cross-linkages||Up to 20 mM|
|DTT, DTE||Low concentrations will reduce nickel ions||A maximum of 1 mM may be reduce nickel ions used, but beta-mercaptoethanol is recommended|
|Nonionic detergents (Triton, Tween, NP-40, etc.)||Removes background proteins and nucleic acids||Up to 2% can be used|
|Cationic detergents||Up to 1% can be used|
|CHAPS||Up to 1% can be used|
|Anionic detergents (SDS, sarkosyl)||Not recommended, but up to 0.3% has been used success-fully in some cases|
|GuHCl||Up to 6 M|
|Urea||Up to 8 M|
|Histidine||Binds to Ni-NTA and competes with histidine residues in the 6xHis tag||Can be used at low concentrations (20 mM) to inhibit non-specific binding and, at higher concentrations (>100 mM), to elute the 6xHis-tagged protein from the Ni-NTA matrix|
|NaCl||Prevents ionic interactions||Up to 2 M can be used, at least 300 mM should be used|
|MgCl2||Up to 4 M|
|CaCl2||Up to 5 mM|
|Glycerol||Prevents hydrophobic interaction between proteins||Up to 50%|
|Ethanol||Prevents hydrophobic interactions between proteins||Up to 20%|
|Imidazole||Binds to Ni-NTA and competes with histidine residues in the 6xHis tag||Can be used at low concentrations (20 mM) to inhibit non-specific binding and, at higher concentrations (>100 mM), to elute the 6xHis-tagged|
|Sodium bicarbonate||Not recommended|
Up to 60mM has been used successfully
Yes, please follow either of the QIAGEN Supplementary Protocols:
Nonionic detergents such as Triton X-100 (0.1 - 1%) and Tween 20 (up to 2%) can be used to reduce non-specific binding of contaminating proteins due to non-specific hydrophobic or ionic interactions. They will have no effect on the binding of 6xHis-tagged protein to the Ni-NTA resin when used within the recommended concentration range.
Optimal concentrations for these additives to binding and wash buffers should be determined empirically for each purification protocol and protein.
To optimize the expression of a given recombinant protein, a time-course analysis of the level of protein expression in the induced culture is recommended. Intracellular protein content is often a balance between the amount of soluble protein in the cells, the formation of inclusion bodies, and protein degradation. By checking the 6xHis-tagged protein present at various times after induction in the soluble and insoluble fractions, the optimal induction period can be established, and the bacterial culture can be harvested at this time. It may be useful to perform plasmid Mini preparations on culture samples during the time-course to enable monitoring of plasmid (expression construct) maintenance.
Below, you can see an example of a time course of recombinant protein expression using the QIAexpress System. You can find this information also in the Section 'Expression in E. coli' in the QIAexpressionist Handbook. The handbook is an important resource for useful background information and protocols. For instructions on how to isolate protein from the soluble and insoluble fractions of induced cultures please see Protocol 14. "Protein minipreps of 6x His-tagged proteins from E. coli under native conditions" and Protocol 19. "6xHis-tagged protein minipreps under denaturing conditions."
Time course of expression using the QIAexpress System. Expression of 6xHis-tagged DHFR was induced with 1 mM IPTG. Aliquots were removed at the times indicated and purified on Ni-NTA Agarose under denaturing conditions. Proteins were visualized by Coomassie staining. Yields per liter culture were 2.8, 5.5,12.3, 33.8, and 53.9 mg, respectively. ■A Crude cell lysate; ■B purification with Ni-NTA. 1: flow-through, 2 & 3: first and second eluates; M: markers; C: noninduced control.