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

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

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02-03-2013, 10:13 AM

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

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

26 October 2012
Publication year: 2012
Source:Biochemical and Biophysical Research Communications, Volume 427, Issue 3

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.
Highlights

? Bile acid binding proteins from different constructs retain structural integrity. ? NMR 15N-T1 relaxation data of BABPs show differences if LVPR extension is present. ? Deviations from a 15N-T1/molecular-weight calibration curve indicate aggregation.

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