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

Ullah R, Shah MA, Tufail S, Ismat F, Imran M, Iqbal M, Mirza O, Rhaman M - PLoS ONE (2016)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Drug Discovery and Structural Biology group, Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

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.‘‘C” represents the cleavable fusion protein treated with protease in the absence of any additive.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4836831&req=5

pone.0153436.g003: 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.‘‘C” represents the cleavable fusion protein treated with protease in the absence of any additive.

Mentions: To further assess the wide applicability of HRV 3C protease, we tested the effect of the buffer composition on the activity. HRV 3C protease cleaved His8-MBP-HRV 3C-MTD efficiently upon 4 hr of incubation at 4°C in each of the four standard buffers used for the elution of polyHis-, GST-, MBP- and Strep-tagged fusion proteins [1, 20–23] (Fig 3). This indicates the robustness of the HRV 3C cleavage protein in a variety of buffers; hence eliminating the time consuming process of desalting or dialysis. Furthermore, rapid cleavage by HRV 3C protease limits compromising target protein integrity. The broad cleavage efficiency of HRV 3C protease identifies it as the protease of choice. In cases where the target protease cleavable protein is unstable in the elution buffer (for example, due to a high imidazole concentration in purifications using Ni-NTA technology), exchange into a suitable buffer by dialysis or any other procedure may be done before cleavage is performed.


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

Ullah R, Shah MA, Tufail S, Ismat F, Imran M, Iqbal M, Mirza O, Rhaman M - PLoS ONE (2016)

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.‘‘C” represents the cleavable fusion protein treated with protease in the absence of any additive.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836831&req=5

pone.0153436.g003: 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.‘‘C” represents the cleavable fusion protein treated with protease in the absence of any additive.
Mentions: To further assess the wide applicability of HRV 3C protease, we tested the effect of the buffer composition on the activity. HRV 3C protease cleaved His8-MBP-HRV 3C-MTD efficiently upon 4 hr of incubation at 4°C in each of the four standard buffers used for the elution of polyHis-, GST-, MBP- and Strep-tagged fusion proteins [1, 20–23] (Fig 3). This indicates the robustness of the HRV 3C cleavage protein in a variety of buffers; hence eliminating the time consuming process of desalting or dialysis. Furthermore, rapid cleavage by HRV 3C protease limits compromising target protein integrity. The broad cleavage efficiency of HRV 3C protease identifies it as the protease of choice. In cases where the target protease cleavable protein is unstable in the elution buffer (for example, due to a high imidazole concentration in purifications using Ni-NTA technology), exchange into a suitable buffer by dialysis or any other procedure may be done before cleavage is performed.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Drug Discovery and Structural Biology group, Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus