Correction: Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production.

[This corrects the article DOI: 10.1371/journal.pone.0153436.]

Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production.

Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications

Expression conditions used for proteins expression.

<p>Expression conditions used for proteins expression.</p

Effect of detergents on the activity of the HRV 3C protease activity.

<p>Effect of detergents on the activity of the HRV 3C protease activity.</p

SDS-PAGE showing the HRV 3C protease activity in the presence various buffers, increasing concentrations of variety of salts, additives and detergents commonly used during extraction and purification of proteins.

<p>‘‘C” represents the cleavable fusion protein treated with protease in the absence of any additive.</p

Analysis of activities of the HRV 3C and TEV proteases at 25°C towards their target proteins (His8-MBP-HRV 3C-MTD or His8-MBP-TEV-MTD, His₈-MBP-HRV 3C-betaC1 or His₈-MBP-TEV-beta C1 and His₈-HRV 3C-100K or His₈-TEV-100K).

<p>Respective cleavable fusion proteins were incubated with HRV 3C protease or TEV protease at ratio of 50:1 for 1 hour and analysed by SDS-PAGE. ‘‘U” and ‘‘T” represent the cleavable fusion protein untreated and treated, respectively, with the HRV 3C or TEV protease, Note: in case of cleavage of 100K fusion protein, 2kDa band was not observed because of low %age of polyacrylamide used in SDS-PAGE.</p

List of oligonucleotides used for cloning.

<p>List of oligonucleotides used for cloning.</p