Protease K Protection Assay: Cell Free Expression Application

Microsomal vesicles are used to study cotranslational and initial posttranslational processing of proteins. Processing events such as signal peptide cleavage, membrane insertion, translocation and core glycosylation can be examined by the transcription/translation of the appropriate DNA in the TNT® Lysate Systems when used with microsomal membranes.

The most general assay for translocation makes use of the protection afforded the translocated domain by the lipid bilayer of the microsomal membrane. In this assay protein domains are judged to be translocated if they are observed to be protected from exogenously added protease. To confirm that protection is due to the lipid bilayer addition of 0.1% non-ionic detergent (such as Triton® X-100) solubilizes the membrane and restores susceptibility to the protease.

Many proteases have proven useful for monitoring translocation in this fashion including Protease K or Trypsin.

The following are examples illustrating this application:

  1. Minn, I. et al. (2009) SUN-1 and ZYG-12, mediators of centrosome-nucleus attachment, are a functional SUN/KASH pair in Caenorhabditis elegans. Mol. Biol. Cell. 20, 4586–95.
  2. Padhan, K. et al. (2007) Severe acute respiratory syndrome coronavirus Orf3a protein interacts with caveolin. J.Gen.Virol. 88, 3067–77.
  3. Tews, B.A. et al. (2007) The pestivirus glycoprotein Erns is anchored in plane in the membrane via an amphipathic helix. J.Biol.Chem. 282, 32730–41.
  4. Pidasheva, S. et al. (2005) Impaired cotranslational processing of the calcium-sensing receptor due to signal peptide missense mutations in familial hypocalciuric hypercalcemia. Hum. Mol. Gen. 14, 1679–90.
  5. Smith, D. et al. (2002) Exogenous peptides delivered by ricin require processing by signal peptidase for transporter associated with antigen processing-independent MHC class I-restricted presentation. J. Immun. 169, 99–107.
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    Gary Kobs

    Strategic Marketing Manager at Promega Corporation
    Gary earned his B.S. in Bacteriology, UW-Madison in 1982. From 1982–1986 he served as Research Tech at UW-Madison. From 1986 to the present Gary has been with Promega Corporation serving in many capacities including as the very first editor of Promega Notes. He was also Manager Tech Services and Training, Product Manager Restriction/Modifying Enzymes, Product Manager Protein Analysis, and is now Marketing Manager Protein Analysis.

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