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Title: Recombinant proteins incorporating short non-native extensions may display increased aggregation propensity as detected by high resolution NMR spectroscopy

Abstract

Highlights: Black-Right-Pointing-Pointer Bile acid binding proteins from different constructs retain structural integrity. Black-Right-Pointing-Pointer NMR {sup 15}N-T{sub 1} relaxation data of BABPs show differences if LVPR extension is present. Black-Right-Pointing-Pointer Deviations from a {sup 15}N-T{sub 1}/molecular-weight calibration curve indicate aggregation. -- Abstract: The use of a recombinant protein to investigate the function of the native molecule requires that the former be obtained with the same amino acid sequence as the template. However, in many cases few additional residues are artificially introduced for cloning or purification purposes, possibly resulting in altered physico-chemical properties that may escape routine characterization. For example, increased aggregation propensity without visible protein precipitation is hardly detected by most analytical techniques but its investigation may be of great importance for optimizing the yield of recombinant protein production in biotechnological and structural biology applications. In this work we show that bile acid binding proteins incorporating the common C-terminal LeuValProArg extension display different hydrodynamic properties from those of the corresponding molecules without such additional amino acids. The proteins were produced enriched in nitrogen-15 for analysis via heteronuclear NMR spectroscopy. Residue-specific spin relaxation rates were measured and related to rotational tumbling time and molecular size. While the native-like recombinant proteins show spin-relaxationmore » rates in agreement with those expected for monomeric globular proteins of their mass, our data indicate the presence of larger adducts for samples of proteins with very short amino acid extensions. The used approach is proposed as a further screening method for the quality assessment of biotechnological protein products.« less

Authors:
; ;  [1];  [1]
  1. Department of Biotechnology, University of Verona, 37134 Verona (Italy)
Publication Date:
OSTI Identifier:
22210313
Resource Type:
Journal Article
Journal Name:
Biochemical and Biophysical Research Communications
Additional Journal Information:
Journal Volume: 427; Journal Issue: 3; Other Information: Copyright (c) 2012 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 0006-291X
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; AMINO ACID SEQUENCE; AMINO ACIDS; BILE ACIDS; BIOTECHNOLOGY; CHEMICAL PROPERTIES; CLONING; MOLECULAR WEIGHT; NITROGEN 15; NUCLEAR MAGNETIC RESONANCE; OPTIMIZATION; PRECIPITATION; PROTEINS; PURIFICATION; RELAXATION; RESIDUES; SPECTROSCOPY; SPIN

Citation Formats

Zanzoni, Serena, D'Onofrio, Mariapina, Molinari, Henriette, and Assfalg, Michael. Recombinant proteins incorporating short non-native extensions may display increased aggregation propensity as detected by high resolution NMR spectroscopy. United States: N. p., 2012. Web. doi:10.1016/J.BBRC.2012.09.121.
Zanzoni, Serena, D'Onofrio, Mariapina, Molinari, Henriette, & Assfalg, Michael. Recombinant proteins incorporating short non-native extensions may display increased aggregation propensity as detected by high resolution NMR spectroscopy. United States. https://doi.org/10.1016/J.BBRC.2012.09.121
Zanzoni, Serena, D'Onofrio, Mariapina, Molinari, Henriette, and Assfalg, Michael. 2012. "Recombinant proteins incorporating short non-native extensions may display increased aggregation propensity as detected by high resolution NMR spectroscopy". United States. https://doi.org/10.1016/J.BBRC.2012.09.121.
@article{osti_22210313,
title = {Recombinant proteins incorporating short non-native extensions may display increased aggregation propensity as detected by high resolution NMR spectroscopy},
author = {Zanzoni, Serena and D'Onofrio, Mariapina and Molinari, Henriette and Assfalg, Michael},
abstractNote = {Highlights: Black-Right-Pointing-Pointer Bile acid binding proteins from different constructs retain structural integrity. Black-Right-Pointing-Pointer NMR {sup 15}N-T{sub 1} relaxation data of BABPs show differences if LVPR extension is present. Black-Right-Pointing-Pointer Deviations from a {sup 15}N-T{sub 1}/molecular-weight calibration curve indicate aggregation. -- Abstract: The use of a recombinant protein to investigate the function of the native molecule requires that the former be obtained with the same amino acid sequence as the template. However, in many cases few additional residues are artificially introduced for cloning or purification purposes, possibly resulting in altered physico-chemical properties that may escape routine characterization. For example, increased aggregation propensity without visible protein precipitation is hardly detected by most analytical techniques but its investigation may be of great importance for optimizing the yield of recombinant protein production in biotechnological and structural biology applications. In this work we show that bile acid binding proteins incorporating the common C-terminal LeuValProArg extension display different hydrodynamic properties from those of the corresponding molecules without such additional amino acids. The proteins were produced enriched in nitrogen-15 for analysis via heteronuclear NMR spectroscopy. Residue-specific spin relaxation rates were measured and related to rotational tumbling time and molecular size. While the native-like recombinant proteins show spin-relaxation rates in agreement with those expected for monomeric globular proteins of their mass, our data indicate the presence of larger adducts for samples of proteins with very short amino acid extensions. The used approach is proposed as a further screening method for the quality assessment of biotechnological protein products.},
doi = {10.1016/J.BBRC.2012.09.121},
url = {https://www.osti.gov/biblio/22210313}, journal = {Biochemical and Biophysical Research Communications},
issn = {0006-291X},
number = 3,
volume = 427,
place = {United States},
year = {Fri Oct 26 00:00:00 EDT 2012},
month = {Fri Oct 26 00:00:00 EDT 2012}
}