In vitro production of N-degron fused proteins and its application

Bong Heon Kim, Do Hoon Kwon, Leehyeon Kim, Woo Seok Yang, Hyun Kyu Song

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

The N-degron pathway, first discovered several decades ago by Varshavsky's laboratory, controls the half-life of target proteins depending on their N-terminal residues. In vivo cell biology studies have established the physiological role of the N-degron pathway. However, in vitro studies such as biochemical assays and structural biology studies are relatively limited. The N-degron substrates cannot be obtained via simple protein expression. The N-degron residues are exposed via the proteolytic process from the translated nascent polypeptide chains. Thus, methods for the fusion expression with several cleavable tags and subsequent treatment with specific proteases to design the exposed N-degron signals have been introduced. Recently, we developed a unique fusion technique using microtubule-associated protein 1A/1B light chain 3B (LC3B), a key marker protein of autophagy, to obtain a high yield of the purified target proteins with variable N-terminal residues for various biochemical studies including enzymatic and binding assays, and crystallization of N-degron complex. This chapter describes the protocols that include the vector map designed for producing LC3B fused target proteins, methods for expression and purification of an example protein, p62/SQSMT1, using different N-terminal residues, and methods to obtain the purified ATG4B protease, which is used for processing LC3B tag and exposing the required N-terminal residues of the target protein.

Original languageEnglish
Title of host publicationModifications and targeting of protein termini
Subtitle of host publicationPart B
EditorsThomas Arnesen
PublisherAcademic Press Inc.
Pages99-123
Number of pages25
ISBN (Print)9780443221002
DOIs
Publication statusPublished - 2023 Jan

Publication series

NameMethods in Enzymology
Volume686
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Bibliographical note

Publisher Copyright:
© 2023 Elsevier Inc.

Keywords

  • ATG4B
  • Crystallization
  • LC3B-fusion
  • N-degron
  • N-end rule
  • N-recognin
  • ZZ-domain
  • p62/SQSTM1

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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