QIAexpress Protein Purification System

The QIAexpress Protein Purification System is designed for one-step purification of 6xHis-tagged proteins by metal-chelate affinity chromatography. Ni-NTA matrices are available for all scales of purification and a wide range of throughput needs. Purification and assay protocols can also be automated on BioRobot Systems.

Features and benefits

• One-step purification from crude lysate to >95% pure protein
• High binding affinity and high capacity
• Choice of purification under native  or denaturing conditions
• Precharged, ready-to-use matrices for any scale of purification
• Automated purification and assay protocols

* For a more complete list of specifications, see table "Ni-NTA Matrices — Specifications".

Principle

The QIAexpress Ni-NTA Protein Purification System is based on the remarkable selectivity of patented Ni-NTA (nickel-nitrilotriacetic acid) resin for proteins containing an affinity tag of six consecutive histidine residues — the 6xHis tag. This technology allows one-step purification of almost any His-tagged protein from any expression system under native or denaturing conditions. NTA, which has four chelation sites for nickel ions, binds nickel more tightly than metal-chelating purification systems that only have three sites available for interaction with metal ions. The extra chelation site prevents nickel-ion leaching and results in a greater binding capacity and protein preparations with higher purity (see figure "One-Step Purification under Native Conditions") than those obtained using other metal-chelating purification systems. The QIAexpress system can be used to purify His-tagged proteins from any expression system including baculovirus, mammalian cells, yeast, and bacteria.

Procedure

The purification of 6xHis-tagged proteins consists of 4 steps: cell lysis, binding, washing, and elution (see flowchart). Purification of recombinant proteins using the QIAexpress system does not depend on the 3-dimensional structure of the protein or 6xHis tag. This allows one-step protein purification under either native or denaturing conditions, from dilute solutions and crude lysates. Strong denaturants and detergents can be used for efficient solubilization and purification of receptors, membrane proteins, and proteins that form inclusion bodies. Reagents that allow efficient removal of nonspecifically binding contaminants can be included in wash buffers (see table "Reagents compatible with the Ni-NTA–6xHis interaction"). Purified proteins are eluted under mild conditions by adding 100–250 mM imidazole as competitor or by a reduction in pH.

Reagents compatible with the Ni-NTA–6xHis interaction*

* The reagents listed have been successfully used in concentrations up to those given.

Applications

The QIAexpress Ni-NTA Protein Purification System provides reliable, one-step purification of proteins suitable for any application, including:

• Structural and functional investigations
• Crystallization for determination of three-dimensional structure
• Assays involving protein–protein and protein–DNA interactions
• Immunization to produce antibodies

Ni-NTA matrices can also be used to bind 6xHis-tagged proteins as immobilized affinity ligands to:

• Study molecular interactions with nucleic acids and binding proteins
• Purify antibodies
• Isolate nontagged, interacting subunits or nucleic acids
• Investigate ligand–receptor interactions

For a list of references citing QIAexpress products for purification of 6xHis-tagged proteins, visit the QIAGEN Reference Database online at www.qiagen.com/RefDB/seach.asp or contact QIAGEN Technical services or your local distributor.

Ni-NTA Matrices — Specifications
	Ni-NTA Agarose	Ni-NTA Superflow	Ni-NTA Spin Columns	Ni-NTA Magnetic Agarose Beads
Gravity-flow column	YES	YES	no	no
FPLC	no	YES	no	no
Scale	Small to medium	Small to production	Microspin minipreps	Micro-scale purifications
Binding capacity	5–10 mg/ml (300–500 nmol @ ~20 kDa)	5–10 mg/ml (300–500 nmol @ ~20 kDa)	150 µg/spin column (7.5 nmol @ ~20 kDa)	300 µg/ml suspension (5%)
Support	Sepharose CL-6B	Superflow	Macroporous silica	Magnetic agarose beads
Bead structure	Cross-linked, 6% agarose	Highly cross-linked, 6% agarose	Silica particles	Cross-linked, 3% agarose
Bead size	45–165 µm	60–160 µm	16–24 µm	Average 50 µm (Range: 20–70 µm)
Exclusion limit (MW)	>>4 x 107	4 x 106	n.d.	n.d.
Max. linear flow rate	75–150 cm/h	3000 cm/h	n.d.	n.a.
Max. volumetric flow rate	0.5–1.0 ml/min	20 ml/min	n.d.	n.a.
Recommended flow rate	0.5 ml/min	1–3 ml/min	0.3 ml/min	n.a.
Max. pressure	2.8 psi (0.2 bar)	140 psi (10 bar)	n.d.	n.a.
pH stability (≤2 h)	2–14	2–14	2–8.5	3–14
pH stability (≥2 h)	3–12	3–12	3–7.5	4–12
Form	50% suspension in 30% ethanol, precharged with Ni2+	50% suspension in 30% ethanol, precharged with Ni2+	Dry matrix packed in spin columns, precharged with Ni2+	5% suspension in 30% ethanol, precharged with Ni2+
Antimicrobial agent	30% ethanol	30% ethanol	n.a.	30% ethanol
Storage	Refrigerated (do not freeze)	Refrigerated (do not freeze)	Refrigerated (do not freeze)	Refrigerated (do not freeze)

n.d.: not determined; n.a.: not applicable