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Recombinant expression of the cardiac troponin I fragment, cTnI[135-209], that controls cardiac contraction

  • Author(s) / Creator(s)
  • Intrinsically disordered regions (IDRs) are protein segments that lack a fixed three-dimensional structure under physiologic conditions, they are susceptible to post-translational modifications such as proteolysis. However, they are highly important in cellular signal transduction and the regulation of numerous biological processes. Cardiac Troponin I (cTnI) is a key regulatory protein in cardiac muscle contraction and relaxation being the inhibitory unit of the troponin complex. Having IDRs, makes it difficult to express. We modified methods to produce human cTnI in E.coli; using fusion partners to direct the target protein to accumulate in dense insoluble aggregates known as inclusion bodies, and protect them from proteolysis, it’s a common approach that’s been widely used to produce many clinically important peptides since the late 1970s, including insulin. CTnI, can be expressed using a PagP protein fusion system developed in our lab. PagP is superior to other fusion systems as it maintains a tandem construct of cTnI fragment in inclusion bodies, and it’s not very hydrophobic, making the inclusion bodies easier to solubilize. We produced cTnI[135-209] segment using PagP fusion system, targeted its expression in E.coli. into inclusion bodies which contained almost pure recombinant protein, solubilized it in denaturant, further purified it using nickel affinity chromatography, and then used Ni assisted hydrolysis to cleave it from PagP, it was isotopically labelled, which allowed us to study it by nuclear magnetic resonance (NMR) spectroscopy to ensure that it’s properly folded and stable. We confirmed that our cTnI[135-209] segment is structurally sound by measuring its molecular weight with MALDI-TOF mass spectrometry, and we ensured that it retained its biological activity by obtaining the chemical shift assignments by NMR before and after titrating actin as we got an evidence of their binding. We plan to use this information to gain a better understanding of heart failure, and cardiomyopathies.

  • Date created
    2015-05-14
  • Subjects / Keywords
  • Type of Item
    Research Material
  • DOI
    https://doi.org/10.7939/R32F7M
  • License
    Attribution-NonCommercial-NoDerivatives 3.0 International