Guidelines to reach high-quality purified recombinant proteins

Abstract

The final goal in recombinant protein production is to obtain high-quality pure protein samples. Indeed, the successful downstream application of a recombinant protein depends on its quality. Besides production, which is conditioned by the host, the quality of a recombinant protein product relies mainly on the purification procedure. Thus, the purification strategy must be carefully designed from the molecular level. On the other hand, the quality control of a protein sample must be performed to ensure its purity, homogeneity and structural conformity, in order to validate the recombinant production and purification process. Therefore, this review aims at providing succinct information on the rational purification design of recombinant proteins produced in Escherichia coli, specifically the tagging purification, as well as on accessible tools for evaluating and optimizing protein quality. The classical techniques for structural protein characterization-denaturing protein gel electrophoresis (SDS-PAGE), size exclusion chromatography (SEC), dynamic light scattering (DLS) and circular dichroism (CD)-are revisited with focus on the protein and their main advantages and disadvantages. Furthermore, methods for determining protein concentration and protein storage are also presented. The guidelines compiled herein will aid preparing pure, soluble and homogeneous functional recombinant proteins from the very beginning of the molecular cloning design.

Quantifying the heterogeneity of macromolecular machines by mass photometry

“…Much effort has therefore been aimed at developing and optimizing techniques capable of accurately reporting on sample heterogeneity including SDS-PAGE, size exclusion chromatography (SEC) and dynamic light scattering (DLS) 8,9 . SDS-PAGE reveals sample size, but not stoichiometry or interactions.…”

Smoothing membrane protein structure determination by initial upstream stage improvements

“…The evaluation of purified protein quality is crucial in any protein production process and should be accurately performed to avoid irreproducible and misleading observations in the subsequent studies (Raynal et al 2014). After production, MP need to be efficiently solubilized (recently reviewed by Hardy et al 2018 andPopot 2018) and purified (Pandey et al 2016), from which their quality in terms of purity, homogeneity, activity, and structural conformity should be assessed (Oliveira and Domingues 2018;Raynal et al 2014). In this review, generic guidelines and host characteristics aiming an accurate choice of the host expression system that better suits particular needs will be initially overviewed in this review, and then we discuss important advances reported at the level of the upstream stage of recombinant MP production processes using E. coli, P. pastoris, and mammalian cell lines, representative of major expression systems used for protein expression.…”

“…The purity and integrity of purified protein samples are usually evaluated by electrophoresis (native or denaturant) coupled with detection methods with varying sensitivities (Oliveira and Domingues 2018;Raynal et al 2014). On the other hand, isoelectric focusing and capillary electrophoresis have also been used to distinguish the protein of interest from closely related undesired subproducts or contaminants (Raynal et al 2014), while UV-Visible spectroscopy is useful to detect nucleic acid contamination (Oliveira and Domingues 2018).…”

“…Dynamic light scattering (DLS) and Fig. 2 Schematic diagram of MP structure determination pipeline focusing relevant parameters to optimize their upstream stage and techniques used to protein quality control more accurately analytical size exclusion chromatography (SEC) are useful to these determinations (Oliveira and Domingues 2018;Raynal et al 2014). In quality control methodologies, studying the secondary and tertiary structure of proteins is important to infer about their folding and monitor protein conformational changes.…”

“…In quality control methodologies, studying the secondary and tertiary structure of proteins is important to infer about their folding and monitor protein conformational changes. A range of spectroscopic techniques has been developed for such task, being circular dichroism particularly useful to determine the secondary structures and folding properties of recombinant proteins (Oliveira and Domingues 2018). Based on several generic or protein-specific functional assays which depend upon catalytic and binding properties of the protein of interest, it is also important to determine the activity of the target protein samples (Raynal et al 2014).…”