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 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 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, 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 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
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

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

(a)

(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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