Protein Purification

Protein purification employs multiple chromatography techniques that separate products according to differences of their properties. Tagged proteins are convenient to be handled by affinity chromatography, which is designed to capture the target protein based on biorecognition of the protein tag. Other protein purification methods, including ion exchange chromatography, size-exclusion chromatography, and hydrophobic interaction chromatography are available if you are interest in obtaining tag-free proteins or products with high purity.

With state-of-art technical platforms for recombinant protein purification, high quality products tailored to your needs are delivered in a short time frame. We are delight to work with you if you have special requirements or protocols for your projects.

Affinity Chromatography

Affinity chromatography

Affinity chromatography is applied in separating biochemical mixtures based on highly specific interactions. Target protein with well-defined property can be exploited in the purification process.

Ion Exchange Chromatography

Ion Exchange Chromatography

Ion exchange chromatography works as a common protein purification method that separates ions and polar molecules based on their affinity to the ion exchanger. Soluble molecules bind to oppositely charged insoluble stationary phase while passing through the column.

Size exclusion chromatography

Size exclusion chromatography

Size exclusion chromatography, also known as gel filtration chromatography, separates molecules by their sizes and molecular weight. Small molecules are trapped in the absorbent materials while larger molecules simply pass by the pores.

Hydrophobic interaction chromatography

Hydrophobic interaction chromatography

Hydrophobic groups are attached to the stationary column. Target proteins with hydrophobic amino acid side chains on surfaces interact with the hydrophobic groups and bind to them in protein purification process.

Selection guide of protein purification methods

Protein purification techniques Affinity chromatography Ion exchange chromatography Size-exclusion chromatography Hydrophobic interaction chromatography
name Affinity chromatography Ion exchange chromatography Size-exclusion chromatography Hydrophobic interaction chromatography
Protein property Biorecognition Charge Size Hydrophobicity
Applications Receptor and ligand, enzyme and substrate, antigen and antibody Charged molecules Large molecules, macromolecular complexes Proteins and peptides with hydrophobic amino acid side chains on surfaces
Advantages
  • Able to isolate one specific protein at a time
  • High recovery yield
  • Rapid separation
  • High accuracy and precision
  • High matrix tolerance
  • High selectivity
  • High recovery yield
  • Well defined separation time
  • Narrow bands available
  • High selectivity
  • Mild, non-denaturing conditions
  • Complementary to ion exchange chromatography and size-exclusion chromatography
Disadvantages Demand ligand with high selectivity Inconsistency from column to column Demand differences in MW Too strong interactions

 

Endotoxin removal in protein purification process

Endotoxin removal is one of the most challenging tasks in the process of protein purification. Multiple techniques are frequently employed to remove endotoxins from protein solutions regard to specific properties of endotoxins in aqueous solution.

Methods Key features
Affinity chromatography High selectivity, highly specific and efficient endotoxin removal combined with excellent target protein recovery
Ion exchange chromatography Rapid separation, wide selection of AEC media, sodium hydroxide (NaOH) sanitization, and solvents-free
Hydrophobic chromatography Interacts with non-polar protein surfaces by van der Waals forces
Size-exclusive chromatography Uses highly porous composite polyacrylamide as the column

Technical Strategies Removal In Protein Purification Process

One-step and automated protein purification

One-step and automated protein purification

Magnetic agarose beads are considered as one of the optimized strategies for one-step and automated protein purification process. Target protein irreversibly binds to the beads and enables efficient protein capture. The strategy eliminates protein loss in of wash steps and results in high protein recovery.

Combination of protein purification strategies

Single step protein purification is frequently insufficient to achieve desired level of purity. The choices of protein purification methods depend greatly upon products properties. Combinations of several classical protein purification techniques are also employed to achieve request purity of proteins without affinity tags.

One-step and automated protein purification
Protein purification methods Capture Intermediate Polish
Affinity chromatography +++ +++ ++
Ion exchange chromatography +++ +++ +++
Size-exclusion chromatography + + +++
Hydrophobic Interaction Chromatography ++ +++ +

Notes:

  • Combine protein purification techniques that apply very different separation mechanisms.
  • Keep balance between purity and yield

 

Trouble shooting guide of protein purification

Problems Possible causes Recommendation
Target protein not bound to the column Protein degradation or tag loss
  • Perform protein purification at low temperature
  • Add protease inhibitors
Tag inaccessible (unexposed)
  • Purify in denaturing conditions
  • Add the tag in other site
Unoptimized binding conditions
  • Change the buffer or binding pH
  • Reduce concentration of imidazole in affinity chromatography
Poor target protein elution Too mild elution conditions
  • Increase imidazole concentration
  • Reduce pH
Too strong interaction with the column
  • Elute with a chelating agent
  • Lower reducing pH in the presence of imidazole
Precipitated target protein Add solubilizing agents
Presence of non-specific hydrophobic or other interactions Change imidazole concentration in gradient elution
High amount of co-eluted contaminants Unoptimized binding conditions
  • Check pH
  • Add saline concentration in the binding buffer
  • Add low concentrations of non-ionic detergents
High concentrated or viscosity sample
  • Make a previous dilution before protein purification
  • Increase sonication time
Insufficient wash
  • Increase washing buffer volume
  • Add low concentration of imidazole in the buffer
Multiple bands observed after WB Partial degradation Add protease inhibitors
Associated contaminants
  • Increase detergent levels
  • Add an additional protein purification step
Cell disruption
  • Decrease lysis time
  • Add lysozyme
Gray or brown resin Presence of reducing agents
  • Remove reducing agents such as DTT
  • Regenerate the resin
Column clogged Presence of cell debris Centrifuge or filter the sample
Protein precipitation
  • Check pH, ionic strength, and protein concentration
  • Use harsh detergent to denature the protein
Inclusion body formation Please see flexible refolding strategies

 

The commonly used FDA-approved techniques for endotoxin detection are the rabbit pyrogen test and Limulus Amoebocyte Lysate (LAL) assay.

Tag Size Matrix Elution condition
His-tag 6-10 His Ni2+-NTA, Co2+-CMA Imidazole 20–250 mM or low pH
Poly-Arg 5-6 Arg Cation-exchange resin NaCl linear gradient from 0 to 400 mM at alkaline pH>8.0
GST (Glutathione-S-transferase) 211 aa Glutathione 5–10 mM reduced glutathione
MBP (maltose-binding protein) 396 aa Cross-linked amylose 10 mM maltose
S-tag 15 aa S-fragment of RNaseA 3 M guanidine thiocyanate
FLAG tag 8 aa Anti-FLAG monoclonal antibody pH 3.0 or 2–5 mM EDTA
Strep-tag 8 aa Strep-Tactin (modified streptavidin) 2.5 mM desthiobiotin
c-myc-tag 11 aa Monoclonal antibody Low pH
Cellulose-binding domain 27–189 aa Cellulose Family I: guanidine HCl or urea>4 M
Family II/III: ethylene glycol
Calmodulin-binding peptide 26 aa Calmodulin EGTA or EGTA with 1 M NaCl

 

Case study of flexible protein purification strategies

Affinity Chromatography(a)
Affinity Chromatography(b)

Comparison of SDS-PAGE analysis of target protein purification

(a) Protein purification using affinity chromatography
Lane M: Protein marker
Lane 1: Supernatant after centrifugation
Lane 2: Flow through
Lane 3-7: 50 mM imidazole eluted fractions
Lane 8-10: 100 nM imidazole eluted fractions
Lane 11-12: 300 nM imidazole eluted fractions

(b) Protein purification using size exclusion chromatography
Lane M: Protein marker
Lane 1: Supernatant after centrifugation
Lane 2-20: Flow through

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