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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

Analysis of activity of the HRV 3C protease and the TEV protease towards their target proteins (His8-MBP-HRV 3C-MTD, His8-MBP-TEV-MTD, His8-MBP-HRV 3C-betaC1, His8-MBP-TEV-beta C1, His8-HRV 3C-100K and His8-TEV-100K) at 25°C and 4°C.The HRV 3C protease or the TEV protease was incubated with respective cleavable fusion protein at ratio of 1:50 and aliquots were taken at the indicated time points and analysed by SDS-PAGE. M denotes the molecular marker while ‘‘C” represents the cleavable fusion protein without any of protease treatment.
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pone.0153436.g002: Analysis of activity of the HRV 3C protease and the TEV protease towards their target proteins (His8-MBP-HRV 3C-MTD, His8-MBP-TEV-MTD, His8-MBP-HRV 3C-betaC1, His8-MBP-TEV-beta C1, His8-HRV 3C-100K and His8-TEV-100K) at 25°C and 4°C.The HRV 3C protease or the TEV protease was incubated with respective cleavable fusion protein at ratio of 1:50 and aliquots were taken at the indicated time points and analysed by SDS-PAGE. M denotes the molecular marker while ‘‘C” represents the cleavable fusion protein without any of protease treatment.

Mentions: We compared the activities of the HRV 3C and TEV proteases towards their three target proteins at 25°C and 4°C. Fig 2 shows that, whilst TEV protease efficiently cleaves the target proteins except His8-TEV-100K after prolonged incubation at 25°C, it was inefficient to do so even after 16 hr at 4°C. In contrast, HRV 3C protease efficiently cleaved all the three fusion proteins after 4 hr at both tested temperatures. Since many recombinant proteins require handling at 4°C, starting from protein purification through to downstream processing, the superior cleavage properties of HRV 3C protease over TEV protease demonstrate the potential to use HRV 3C protease for recombinant protein production [19]. In addition, HRV 3C protease should also be the protease of choice at 25°C, as our experiments show that its activity at higher temperatures is also superior to that of TEV protease (Fig 2).


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)

Analysis of activity of the HRV 3C protease and the TEV protease towards their target proteins (His8-MBP-HRV 3C-MTD, His8-MBP-TEV-MTD, His8-MBP-HRV 3C-betaC1, His8-MBP-TEV-beta C1, His8-HRV 3C-100K and His8-TEV-100K) at 25°C and 4°C.The HRV 3C protease or the TEV protease was incubated with respective cleavable fusion protein at ratio of 1:50 and aliquots were taken at the indicated time points and analysed by SDS-PAGE. M denotes the molecular marker while ‘‘C” represents the cleavable fusion protein without any of protease treatment.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153436.g002: Analysis of activity of the HRV 3C protease and the TEV protease towards their target proteins (His8-MBP-HRV 3C-MTD, His8-MBP-TEV-MTD, His8-MBP-HRV 3C-betaC1, His8-MBP-TEV-beta C1, His8-HRV 3C-100K and His8-TEV-100K) at 25°C and 4°C.The HRV 3C protease or the TEV protease was incubated with respective cleavable fusion protein at ratio of 1:50 and aliquots were taken at the indicated time points and analysed by SDS-PAGE. M denotes the molecular marker while ‘‘C” represents the cleavable fusion protein without any of protease treatment.
Mentions: We compared the activities of the HRV 3C and TEV proteases towards their three target proteins at 25°C and 4°C. Fig 2 shows that, whilst TEV protease efficiently cleaves the target proteins except His8-TEV-100K after prolonged incubation at 25°C, it was inefficient to do so even after 16 hr at 4°C. In contrast, HRV 3C protease efficiently cleaved all the three fusion proteins after 4 hr at both tested temperatures. Since many recombinant proteins require handling at 4°C, starting from protein purification through to downstream processing, the superior cleavage properties of HRV 3C protease over TEV protease demonstrate the potential to use HRV 3C protease for recombinant protein production [19]. In addition, HRV 3C protease should also be the protease of choice at 25°C, as our experiments show that its activity at higher temperatures is also superior to that of TEV protease (Fig 2).

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