Available online 20 September 2022

Soluble and solubilized proteins may show different activities.

The procedure used for protein solubilization determines the protein final activity.

Inclusion bodies can be solubilized without use of solubilizing agents.

Protein activity needs to be monitored after the solubilization process

Recombinant protein production in bacteria is often accompanied by the formation of aggregates, known as inclusion bodies (IBs). Although several strategies have been developed to minimize protein aggregation, many heterologous proteins are produced in aggregated form. For these proteins, purification necessarily requires processes of solubilization and refolding, often involving denaturing agents. However, the presence of biologically active recombinant proteins forming IBs has driven a redefinition of the protocols used to obtain soluble protein avoiding the protein denaturation step. Among the different strategies described, the detergent n-lauroylsarcosine (NLS) has proved to be effective. However, the impact of the NLS on final protein quality has not been evaluated so far. Here, the activity of three antimicrobial proteins (all as GFP fusions) obtained from the soluble fraction was compared with those solubilized from IBs. Results showed that NLS solubilized proteins from IBs efficiently, but that protein activity was impaired. Thus, a solubilization protocol without detergents was evaluated, demonstrating that this strategy efficiently solubilized proteins embedded in IBs while retaining their biological activity. These results showed that the protocol used for IB solubilization has an impact on final protein quality and that IBs can be solubilized through a very simple step, obtaining fully active proteins.

guanidine hydrochloride

Green Fluorescent Protein

glutathione S-transferase

isopropyl β-D-1-thiogalactopyranoside

lingual antimicrobial peptide

maltose-binding protein

Inclusion bodies

protein quality

mild solubilization

n-lauroylsarcosine

soluble protein

solubilized protein

© 2022 Published by Elsevier B.V.