Tag-free Protein Production

Efficient tag removal services offered by BiologicsCorp generate high yields of tag-free protein with desired amino acids. The tag-removal services are often recommended to prevent interference with target protein function or interactions.

Tagged protein vs. tag-free protein

Tagged protein of interest (POI)

Tagged protein of interest (POI)

Tag-free protein of interest (POI)
Tag-free protein of interest (POI)
Protein structure and function Large tag may limit the folding of recombinant proteins and has significant impact on protein structure and biological functions Identical sequences and similar structures with native proteins
Immunogenicity Possible to generate antibodies with non-specific binding Improved specific binding
Crystallization and X-ray structure analysis Exogenous tag amino acids may cause changes of crystal structure and functional domain analysis Close to native protein crystal structure
FDA approval The recombinant protein with exogenous amino acids has limited chance to pass the FDA approval More likely to pass the FDA approval

Tag free protein production description

Content Tag-free protein production
Deliverable
  • Purified protein with guaranteed quantity (3mg, 5mg, 10mg, 15+mg) and purity (85%, 90%, 95%)
  • Cloning vector
  • QC data (SDS-PAGE, MALDI-TOF)
Timeline 5-8 weeks
Price Please inquire
Additional services Guaranteed protein expression service, fermentation service, SupernateIN soluble expression optimization, endotoxin removal, high-throughput protein production

 

Multiple available fusion tag removal methods
Fig 1. Multiple available fusion tag removal methods
  • Tag-free methods: starting gene synthesis without exogenous amino acids is usually considered as a priority. Flexible purification methods will be applied to tag-free protein production.
  • Enzymatic cleavage methods: one of the most conventional methods to carry on fusion tag-removal experiments. We have established a fast and easy-to-manipulate protocol to screen the binding tag and obtain high purity protein linking Magnetic Agarose Beads with the tagged protein of interest.
  • Self-cleaving enzymatic methods: inteins and other self-splicing proteins have the unique ability to excise themselves from a translated host protein.

 

Principle Advantages Disadvantages
Tag-free methods Start gene synthesis without exogenous amino acids
  • No exogenous amino acids or proteases in products
  • Target tag-free protein was obtained in the elution fraction with high homogeneity
Non-affinity purification methods
Enzymatic cleavage methods Cleavage using highly-specific proteases
  • Specific
  • Cleavage achieved usually under mild conditions
  • Leakage of the endoprotease from the column during purification
  • Contaminating proteases derived from the host organism or the endoprotease preparation may contribute to product degradation
  • Relatively high cost
Self-cleaving enzymatic methods Insertion of a self-cleaving enzyme between an affinity tag and the protein enables the elution of the protein from the affinity column,leaving the enzyme and the affinity tag on the column Enable purification,cleavage and target separation to be achieved in a single step The refolding and cleavage processes are concentration-dependent with reduced yields obtained for large proteins

 

Tag Size Matrix Defects
GST (Glutathione-S-transferase) 211 aa Glutathione
  • Degradation problem cause by large size
  • Affinity for glutathione resin depends on certain reagents
MBP (maltose-binding protein) 396 aa Cross-linked amylose
  • Expression of target proteins is sometimes problematic due to large size
S-tag 15 aa S-fragment of RNaseA
  • Elution of the target protein requires cleavage of the tag or harsher denaturing conditions that disrupt the S-tag S-protein interaction
FLAG tag 8 aa Anti-FLAG monoclonal antibody
  • The monoclonal antibody purification matrix is not as stable as others
  • High cost
Strep-tag 8 aa Strep-Tactin (modified streptavidin)
  • Highly variable binding and purification conditions
c-myc-tag 11 aa Monoclonal antibody
  • The washing conditions are physiological followed by elution at low pH, which could exert a negative effect on protein activity
Cellulose-binding domain 27–189 aa Cellulose
  • Low-polarity solvent presumably disrupts the hydrophobic interaction at the binding site.
Calmodulin-binding peptide 26 aa Calmodulin
  • May reduce the efficiency of translation
NusA,TrxA and DsbA NA NA
  • Cannot be purified with a specific affinity matrix

 

Challenges:
  • Target tag-free protein with small MW
  • Similar size of target tag-free protein (~14.7Kd) and impurity protein (~10Kd)
Case Study
Fig 2. Multi-step purification of a tag-free protein production project
Lane M: Protein marker
Lane 1: Protein sample
Lane 2-15: Target protein eluted from the columns (series connection)
Lane 16-23: Impurity band isolated from the target protein

Contact Us

  +1 (317) 703-0614
  +1 (855) 427-1516

* To get a price quotation, please fill the protein service pre-order form and email to Sales@BiologicsCorp.com.