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

Lactoferrin cDNA. Expression and in vitro mutagenesis

J W Tweedie1, H B Bain, C L Day

  • 1Department of Chemistry and Biochemistry, Massey University, Palmerston North, New Zealand.

Advances in Experimental Medicine and Biology
|January 1, 1994
PubMed
Summary

Researchers synthesized full-length human lactoferrin cDNA and expressed it in cells, achieving high yields. They also created and expressed an N-lobe mutant (LfN), which binds iron but releases it at a different pH than the native protein.

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

RYR1 variant c.38T>G, p.Leu13Arg causes hypersensitivity of the ryanodine receptor-1 and is pathogenic for malignant hyperthermia.

British journal of anaesthesia·2021
Same author

The RING Domain of RING Finger 12 Efficiently Builds Degradative Ubiquitin Chains.

Journal of molecular biology·2020
Same author

Nuclear Phosphoinositides: Their Regulation and Roles in Nuclear Functions.

International journal of molecular sciences·2019
Same author

A genetic mystery in malignant hyperthermia 'solved'?

British journal of anaesthesia·2018
Same author

Evidence of malignant hyperthermia in patients administered triggering agents before malignant hyperthermia susceptibility identified: missed opportunities prior to diagnosis.

Anaesthesia and intensive care·2017
Same author

Administration of anaesthetic triggering agents to patients tested malignant hyperthermia normal and their relatives in New Zealand: an update.

Anaesthesia and intensive care·2017

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Protein Engineering

Background:

  • Human lactoferrin is an iron-binding protein with antimicrobial and immune-modulating functions.
  • Understanding lactoferrin's structure-function relationship is crucial for therapeutic applications.

Purpose of the Study:

  • To synthesize and express full-length human lactoferrin and its N-lobe mutant (LfN).
  • To investigate the iron-binding and release properties of the LfN mutant.
  • To engineer mutations in LfN to mimic C-lobe characteristics.

Main Methods:

  • cDNA synthesis using polymerase chain reaction (PCR) and reverse transcriptase.
  • Expression of recombinant proteins in baby hamster kidney (BHK) cells using the pNUT vector.
  • Site-directed mutagenesis to create modified LfN proteins.

Related Experiment Videos

  • Biophysical characterization including UV/Vis and ESR spectroscopy.

    • Main Results:

    • High yields (up to 40 mg/L) of secreted full-length human lactoferrin and LfN were obtained.
    • LfN demonstrated iron-binding capacity with spectral properties similar to native lactoferrin.
    • Iron release from LfN occurred at a different pH compared to native and full-length recombinant lactoferrin.
    • Mutant LfN proteins with altered iron-binding ligands were successfully expressed.

    Conclusions:

    • The study successfully produced recombinant human lactoferrin and a functional N-lobe mutant (LfN) in high yields.
    • LfN exhibits distinct iron release kinetics, suggesting domain-specific roles in iron binding and release.
    • Engineered mutations provide insights into the structural determinants of iron binding and potential therapeutic modifications.