MINI-REVIEW Guidelines to reach high-quality purified recombinant proteins Carla Oliveira 1 & Lucília Domingues 1 Received: 6 September 2017 /Revised: 24 October 2017 /Accepted: 27 October 2017 /Published online: 18 November 2017 # Springer-Verlag GmbH Germany, part of Springer Nature 2017 Abstract The final goal in recombinant protein production is to obtain high-quality pure protein samples. Indeed, the suc- cessful downstream application of a recombinant protein de- pends 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 pro- viding succinct information on the rational purification design of recombinant proteins produced in Escherichia coli, specif- ically 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 chro- matography (SEC), dynamic light scattering (DLS) and circu- lar 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 molecu- lar cloning design. Keywords Recombinant protein . Fusion tags . Protein purification . Structural characterization . Quality control . Protein quantification Introduction Recombinant protein production (RPP) has been increasingly used in laboratorial research for obtaining recombinant pro- teins for biophysical and structural studies (Vedadi et al. 2010), diagnostic and therapeutic purposes (Jozala et al. 2016), as well as emerging applications, such as smart mate- rials (Hollingshead et al. 2017). The RPP field constitutes a multi-billion dollar market since a significant part of the main biotechnological market products are recombinant proteins. Namely, the total market sales from microbial recombinant products have reached approximately $50 billion in 2016, representing one third of the total sales of biopharmaceuticals (Jozala et al. 2016). Regardless the final protein application, high-quality protein samples must be obtained upon the RPP process, which could fulfil established purity and conforma- tional requirements. Purification and characterization of re- combinant proteins can be demanding, expensive and time- consuming, but can determine protein quality. That is, the successful application of a recombinant protein depends, to a great extent, on its efficient downstream processing. This comprises protein purification, quality validation, quantifica- tion and storage. The design of a rational protein purification strategy should be the first step in the overall RPP strategy. A poor purification design may result in misfolded or heteroge- neous protein samples due to the interference of sequence additions, such as tags or extra amino acids resulting from the cloning procedure, and/or absence of refining steps in the purification procedure. Thus, adequate structural studies must be conducted to access the quality of the purified * Lucília Domingues luciliad@deb.uminho.pt 1 CEB - Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal Appl Microbiol Biotechnol (2018) 102:81–92 https://doi.org/10.1007/s00253-017-8623-8