Experimental Validation of the Predictions of a Mathematical Model for Protein Purification and Tag Selection

Abstract

To improve downstream processing, protein engineering can be used to modify particular properties of a protein, such as specific affinity, charge, or hydrophobicity. The most common modification involves the peptide tags fusion to the protein. Nevertheless, the selection of both the optimal peptide tag and the right purification system to use is not trivial. The aim of this work is to experimentally validate our previous mathematical model based on MINLP models. This model was modified to find the minimum number of chromatographic operations, and the ideal tag for obtaining a required level of purification. A weighted linear combination of the number of purification steps and the target protein purity obtained after the last step was used as the objective function. The chromatographic steps suggested by the model were carried out using an example based on two mutated cutinases. The results show that average deviations between experimental data and those predicted by the model, for yield and purity, are ca. 15%, confirming that model predictions are reliable and could be used for selecting the best purification processes without experimental tests.

Keywords:

• cutinases
• experimental validation
• model
• protein purification processes
• tagged proteins

ACKNOWLEDGEMENTS

This work was supported by FONDECYT project 108143, FONDECYT Research Initiation grant 11080016 and the Millennium Scientific Initiative ICM project P05-001-F. Plasmid pFCEX1 was a generous gift of Dr. Evamaria I. Petersen, Dept. of Physics and Nanotechnology, University of Aalborg, Denmark.

Notes

¹σ _i is used for determining reduction of contaminants after applying a chromatographic technique i.

²CF is used for estimating the mass of contaminant p after chromatographic step i has been applied (20).

³DF represents the driving force of the separation process.

⁴It is assumed that the separation occurs with a product loss which depends upon the chromatographic techniques and amino acidic composition of the polypeptide tag.

¹Molecular weight was measured by SDS-PAGE with PhastGel media in Phast System.

²Hydrophobicity calculated using equation (24).

³ is the total surface of the original protein (without tag).

⁴Titration curve of cutinase (PDB Code 1CEX) was obtained using the program http://www.iut-arles.up.univ-mrs.fr/w3bb/d_abim/compo-p.html and for the mutants using Eq. (23).

⁵Hydrophobicity was measured by HIC using a phenyl-superose gel in an FPLC and a gradient elution from 2.0 M to 0.0 M (NH₄)₂SO₄ in 20 mM Tris buffer.

*Charge was measured by electrophoretic titration curve analysis with PhastGel IEF 3–9 in a Phast System.

Underlined triplets correspond to the codons for the hydrophobic amino acids added to the C-terminal of F. solani cutinase.

**Average between difference of yield and difference of purity.

***Average between Average Cutinase_(wp)2 and Average Cutinase_(y)3.