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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 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).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.
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pone.0153436.g001: 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).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.

Mentions: Protease cleavable 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 have calculated molecular masses of ~ 55.2 kDa, ~ 59.2 kDa and ~ 91 kDa, respectively. Molecular masses of proteins were calculated using the ExPASy Bioinformatic Portal, http://web.expasy.org/compute_pi/. All proteins were produced by recombinant means as described in materials and methods. Cleavage of the target fusion proteins with the HRV 3C or TEV protease releases two major protein bands of ~ 45 kDa (His8-MBP) and ~ 10 kDa (MTD), ~ 45 kDa (His8-MBP) and 14 kDa (beta C1), and ~89 kDa (100K) and ~2 kDa (His8), respectively (Fig 1). For clarity, we only show the bands corresponding to the uncut fusion proteins and large size cut fragments of the fusion proteins in subsequence SDS-polyacrylamide gels.


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 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).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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153436.g001: 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).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.
Mentions: Protease cleavable 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 have calculated molecular masses of ~ 55.2 kDa, ~ 59.2 kDa and ~ 91 kDa, respectively. Molecular masses of proteins were calculated using the ExPASy Bioinformatic Portal, http://web.expasy.org/compute_pi/. All proteins were produced by recombinant means as described in materials and methods. Cleavage of the target fusion proteins with the HRV 3C or TEV protease releases two major protein bands of ~ 45 kDa (His8-MBP) and ~ 10 kDa (MTD), ~ 45 kDa (His8-MBP) and 14 kDa (beta C1), and ~89 kDa (100K) and ~2 kDa (His8), respectively (Fig 1). For clarity, we only show the bands corresponding to the uncut fusion proteins and large size cut fragments of the fusion proteins in subsequence SDS-polyacrylamide gels.

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