One-Step Protein Purification

 

Lysate Preparation and One-Step Purification/Chromatography
Starting material can be up to 200g of cell paste/pellet (10-20L of E. coli, 10L of insect cells, or 10L mammalian cells). Cell pellet(s) will be thawed on wet ice, suspended in lysis buffer and disrupted by microfluidization followed by centrifugation (15000g, 4C, 1h) to clarify the crude extract. The clarified lysate will be applied to 1st step chromatographic media, washed (to baseline OD280), and then developed as appropriate for the method. Target protein recovery will be monitored by total protein assay (Bradford procedure) and SDS-PAGE of the eluted fractions. Details are provided below with the particular chromatography method.

Lysis Buffer - Choose buffer and additional components for His-tagged, GST-fusions or ion exchange. All lysis buffers include Protease Inhibitor Cocktail (AEBSF, Aprotinin, Bestatin, E-64, Leupeptin, Pepstatin A), +/- EDTA.

Buffer Salt (0 to 50mM): Any of (i) Tris-HCl (ii) Sodium Phosphate (iii) HEPES.

Additional Components
EDTA (0-2mM)
Sodium Chloride (0-500mM)
Magnesium Chloride (0-5mM)
Glycerol (0-10%)
Reducing Agents: (i) DTT (ii) 2-mercaptoethanol (iii) TCEP 
Detergents: (i) Triton X-100 (ii) Brij 35 (iii) Tween 20 (iv) NP-40
Affinity-related related reagents: (i) imidazole for His-tags (ii) reduced glutathione for GST-fusions

Please Note: Other buffer components not listed here (e.g., buffer salts, special, and high-purity detergents) can be included at supplier cost. Just let us know what you need.

 

One- or First-Step Ni-NTA / His-Tagged Proteins
Lysis buffer will include imidazole (0-20mM). Clarified lysate will be batch bound to 10mL of Ni-NTA Superflow (capacity of NiNTA resin is ~50mg his-tagged protein/mL packed resin), loaded into an FPLC column, and washed to baseline (OD280nm) with lysis buffer (minus protease inhibitors). Further washing with lysis buffer containing a slightly higher concentration of imidazole is an option. Elution of his-tagged protein will be either step or linear gradient of buffer containing up to 300mM imidazole. The potential yield is 500mg using 10mL of NiNTA superflow. Fractions will be analyzed for total protein (Bradford) and purity by SDS-PAGE. Elution profile results (SDS-gel) will be immediately communicated to customer for decisions on pooling of best eluted fractions. Buffer exchange and concentration for additional charge (see below).

Delivered: Purified protein in elution buffer with amount determined by expressed levels in starting material and efficiency of binding to NiNTA.
Time: 4-6 days.

 

One- or First-Step Glutathione Affinity / GST-Fusion Proteins
Clarified lysate will be bound to 10mL of glutathione agarose (NOTE: capacity of glutathione agarose is >8 mg GST-fusion protein/mL resin) and loaded into a column and washed to baseline (OD280nm) with lysis buffer (minus protease inhibitors). Elution of protein will be with lysis buffer containing 10mM reduced glutathione. The potential yield is >80mg using 10mL of glutathione agarose. Fractions will be analyzed for total protein (Bradford) and purity by SDS-PAGE. Elution profile results (SDS-gel) will be sent to customer for decisions on pooling of best eluted fractions. Buffer exchange and concentration for additional charge (see below).

Delivered: Purified protein in elution buffer (10mM glutathione) with final amount determined by expressed levels in starting material and efficiency of binding and elution to glutathione agarose.
Time: 4-6 days.

 

One- or First-Step Ion Exchange Chromatography (DEAE / Q / SP / CM)
Clarified lysate made in a suitable pH and low ionic strength buffer will be passed over a 500mL bed of either Q, DEAE, SP or CM-Sepharose and washed to baseline (OD280nm) with buffer (minus protease inhibitors). Please note that a typical clarified lysate from 100g cells contains ~25g protein and a 500mL of IEX-Sepharose will bind 50 to 100g of protein. Elution of protein will be accomplished by development of column with sodium chloride gradient in lysis buffer. Fractions will be analyzed for total protein (Bradford) and purity by SDS-PAGE. Elution profile results (SDS-gel) will be communicated for decisions on pooling of best fractions.

Delivered: Purified protein in elution buffer (10mM glutathione) with final amount determined by levels expressed in cell material and efficiency of binding and elution with IEX-Sepharose.
Time: 4-6 days.