Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Dihydrofolate reductase as a new "affinity handle".

M Iwakura1, K Furusawa, T Kokubu

  • 1Research Institute for Polymers and Textiles, Ibaraki.

Journal of Biochemistry
|January 1, 1992
PubMed
Summary

Dihydrofolate reductase (DHFR) serves as an effective affinity handle for recombinant protein expression. This method enhances soluble protein production, stabilizes polypeptides, and simplifies purification using methotrexate chromatography.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Effects of a flow field on amyloid fibrillogenesis in a β-lactoglobulin solution.

International journal of biological macromolecules·2014
Same author

Establishment of a new cell line susceptible to Cyprinid herpesvirus 3 (CyHV-3) and possible latency of CyHV-3 by temperature shift in the cells.

Journal of fish diseases·2014
Same author

Nanometric locking of the tight focus for optical microscopy and tip-enhanced microscopy.

Nanotechnology·2012
Same author

Wheelchair half-marathon race increases natural killer cell activity in persons with cervical spinal cord injury.

Spinal cord·2012
Same author

Effect of age on bowel management in traumatic central cord syndrome.

Spinal cord·2011
Same author

Plasma IL-6 levels during arm exercise in persons with spinal cord injury.

Spinal cord·2011

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • Dihydrofolate reductase (DHFR) is a key enzyme in folate metabolism.
  • DHFR can be utilized as an affinity handle for recombinant protein expression and purification.
  • Fusion proteins incorporating DHFR offer advantages in solubility and stability.

Purpose of the Study:

  • To evaluate DHFR as a versatile affinity handle for expressing and purifying recombinant polypeptides.
  • To demonstrate the efficiency of DHFR-mediated purification using methotrexate affinity chromatography.
  • To assess the utility of DHFR fusion proteins for producing specific target polypeptides.

Main Methods:

  • Chemical synthesis of 15 genes encoding polypeptides of varying lengths (5-44 amino acids).

Related Experiment Videos

  • Expression of synthetic genes as DHFR fusion proteins in Escherichia coli.
  • Purification of fusion proteins using methotrexate-bound affinity chromatography, monitoring DHFR activity.
  • Cleavage of purified fusion proteins with cyanogen bromide (BrCN) or site-specific protease.
  • Final purification of target polypeptides via reversed-phase high-pressure liquid chromatography (HPLC).

    • Main Results:

    • Efficient expression of all DHFR fusion proteins in E. coli, exhibiting DHFR activity.
    • High-yield purification of fusion proteins from cell-free extracts.
    • Methotrexate affinity chromatography proved highly effective for purification.
    • Specific cleavage of fusion proteins resulted in minimal peptide fragments.
    • Facilitated assignment and isolation of target polypeptides.

    Conclusions:

    • DHFR is a versatile and efficient affinity handle for recombinant protein expression.
    • DHFR fusion proteins enhance protein solubility, stability, and simplify purification.
    • Methotrexate affinity chromatography combined with specific cleavage is a robust method for isolating target polypeptides.
    • This strategy enables the straightforward production and purification of specific polypeptides.