title: Efficient segmental isotope labeling of multi-domain proteins using Sortase A.
abstract: NMR studies of multi-domain protein complexes provide unique insight into their molecular interactions and dynamics in solution. For large proteins domain-selective isotope labeling is desired to reduce signal overlap, but available methods require extensive optimization and often give poor ligation yields. We present an optimized strategy for segmental labeling of multi-domain proteins using the S. aureus transpeptidase Sortase A. Critical improvements compared to existing protocols are (1) the efficient removal of cleaved peptide fragments by centrifugal filtration and (2) a strategic design of cleavable and non-cleavable affinity tags for purification. Our approach enables routine production of milligram amounts of purified segmentally labeled protein for NMR and other biophysical studies.
authors: Institute of Structural Biology, Helmholtz Zentrum München, 85764, Neuherberg, Germany. lee.freiburger@tum.de.|Center for Integrated Protein Science Munich (CIPSM) at Department of Chemistry, Technische Universität München, Lichtenbergstr.4, 85747, Garching, Germany. lee.freiburger@tum.de.
authors: Lee
authors: L
authors: Freiburger
authors: Institute of Structural Biology, Helmholtz Zentrum München, 85764, Neuherberg, Germany. miriam.sonntag@mytum.de.|Center for Integrated Protein Science Munich (CIPSM) at Department of Chemistry, Technische Universität München, Lichtenbergstr.4, 85747, Garching, Germany. miriam.sonntag@mytum.de.
authors: Miriam
authors: M
authors: Sonntag
authors: Institute of Structural Biology, Helmholtz Zentrum München, 85764, Neuherberg, Germany. janosch.hennig@helmholtz-muenchen.de.|Center for Integrated Protein Science Munich (CIPSM) at Department of Chemistry, Technische Universität München, Lichtenbergstr.4, 85747, Garching, Germany. janosch.hennig@helmholtz-muenchen.de.
authors: Janosch
authors: J
authors: Hennig
authors: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China. lijianzhongbei@163.com.
authors: Jian
authors: J
authors: Li
authors: Institute of Structural Biology, Helmholtz Zentrum München, 85764, Neuherberg, Germany. peijian.zou@helmholtz-muenchen.de.|Center for Integrated Protein Science Munich (CIPSM) at Department of Chemistry, Technische Universität München, Lichtenbergstr.4, 85747, Garching, Germany. peijian.zou@helmholtz-muenchen.de.|Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China. peijian.zou@helmholtz-muenchen.de.
authors: Peijian
authors: P
authors: Zou
authors: Institute of Structural Biology, Helmholtz Zentrum München, 85764, Neuherberg, Germany. sattler@helmholtz-muenchen.de.|Center for Integrated Protein Science Munich (CIPSM) at Department of Chemistry, Technische Universität München, Lichtenbergstr.4, 85747, Garching, Germany. sattler@helmholtz-muenchen.de.|Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China. sattler@helmholtz-muenchen.de.
authors: Michael
authors: M
authors: Sattler
mesh_terms: D019881:Aminoacyltransferases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007553:Isotope Labeling / Q000379:methods*; D017434:Protein Structure, Tertiary; D066244:Proton Magnetic Resonance Spectroscopy; D013211:Staphylococcus aureus / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases
keywords: Multi-domain proteins; Protein expression; Protein ligation; Segmental isotope labeling; Sortase A