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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

SDS-PAGE analysis of cleavage of His8-MBP-HRV 3C-domain by the HRV 3C protease in the presence of various detergents. Each lane is labeled according to detergents serial number as detailed in Table 3.‘‘C” represents the cleavable fusion protein treated with protease in the absence of any detergent.
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pone.0153436.g004: SDS-PAGE analysis of cleavage of His8-MBP-HRV 3C-domain by the HRV 3C protease in the presence of various detergents. Each lane is labeled according to detergents serial number as detailed in Table 3.‘‘C” represents the cleavable fusion protein treated with protease in the absence of any detergent.

Mentions: Finally, we assessed the activity of the HRV 3C protease in the presence of different detergents at 4°C. These chemicals are used for different purposes, including the extraction of hydrophobic proteins from biological membranes, refolding, crystallization and protein stabilization. We digested His8-MBP-HRV 3C-MTD in the presence of various detergents at their critical micelle concentration with HRV 3C protease (Table 3 and Fig 4). Of the forty nine detergents tested, fourteen detergents (ANAPOE®-80, n-Tetradecyl-β-D-maltoside, ANAPOE®-C13E8, C12E8, ANAPOE®-C12E10, Sucrose monolaurate, CYMAL®-6, 2,6-Dimethyl-4-heptyl-β-D-maltopyranoside, NDSB-195, DDMAB, ZWITTERGENT® 3–10, LysoFos™ Choline 10, n-Dodecyl-β-D-maltoside and TRITON® X-100) did not affect the HRV 3C protease activity and the target protein was efficiently cleaved (~ 80 to 100%) compared to a control reaction in absence of any detergent. In the presence of fifteen detergents (n-Undecyl-β-D-maltoside, n-Nonyl-β-D-thioglucoside, CYMAL®-5, n-Nonyl-β-D-maltoside, C8E4, C-HEGA®-11, n-Octyl-β-D-glucoside, FOS-Choline®-12, FOS-Choline®-8 fluorinated, CHAPS, CHAPSO, ANAPOE®-58, CYMAL®-4, Sulfobetaine 3–10 and LDAO) HRV 3C protease activity was reduced to ~ 50% relative to the control reaction and the activity decreased further, below 50%, relative to the cleavage in the control reaction in the presence of another twelve detergents (MEGA-9, HEGA®-9, n-Hexyl-β-D-glucopyranoside, ZWITTERGENT® 3–12, MEGA-10, Pluronic® F-68, HECAMEG®, CYMAL®-3, NDSB-256, FOS-MEA®-10, LysoFos™ Choline 12, and Sodium dodecanoylsarcosine). HRV 3C protease activity was completely abolished in the presence of eight detergents (C-HEGA®-10, C-HEGA®-9, HEGA®-8, CYMAL®-2, n-Decyl-N,N-dimethylglycine, MEGA-8, FOS-Choline®-8 and ANAPOE®-20). In 2011, Vergis and Weiner [4] reported a study on the cleavage activity of HRV 3C protease in the presence of a wide range of detergents at 25°C. Although the substrates are different and half of the detergents tested are different, comparison of both datasets for the same detergents show that, in most cases, HRV 3C protease activity is preserved better at 4°C. For example, whereas Vergis and Weiner [4] report little or no cleavage in the presence of FOS-choline-12, HRV 3C protease cleaves over 50% of the target protein at 4°C. In contrast, cleavage is better at 25°C in the presence of C8E4, HECAMEG and n-undecyl-b-D-maltoside [4]. On the basis of structure of detergents, it has been observed that HRV 3C protease is inhibited to less extent by maltoside and glycol ether containing detergents.


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 analysis of cleavage of His8-MBP-HRV 3C-domain by the HRV 3C protease in the presence of various detergents. Each lane is labeled according to detergents serial number as detailed in Table 3.‘‘C” represents the cleavable fusion protein treated with protease in the absence of any detergent.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153436.g004: SDS-PAGE analysis of cleavage of His8-MBP-HRV 3C-domain by the HRV 3C protease in the presence of various detergents. Each lane is labeled according to detergents serial number as detailed in Table 3.‘‘C” represents the cleavable fusion protein treated with protease in the absence of any detergent.
Mentions: Finally, we assessed the activity of the HRV 3C protease in the presence of different detergents at 4°C. These chemicals are used for different purposes, including the extraction of hydrophobic proteins from biological membranes, refolding, crystallization and protein stabilization. We digested His8-MBP-HRV 3C-MTD in the presence of various detergents at their critical micelle concentration with HRV 3C protease (Table 3 and Fig 4). Of the forty nine detergents tested, fourteen detergents (ANAPOE®-80, n-Tetradecyl-β-D-maltoside, ANAPOE®-C13E8, C12E8, ANAPOE®-C12E10, Sucrose monolaurate, CYMAL®-6, 2,6-Dimethyl-4-heptyl-β-D-maltopyranoside, NDSB-195, DDMAB, ZWITTERGENT® 3–10, LysoFos™ Choline 10, n-Dodecyl-β-D-maltoside and TRITON® X-100) did not affect the HRV 3C protease activity and the target protein was efficiently cleaved (~ 80 to 100%) compared to a control reaction in absence of any detergent. In the presence of fifteen detergents (n-Undecyl-β-D-maltoside, n-Nonyl-β-D-thioglucoside, CYMAL®-5, n-Nonyl-β-D-maltoside, C8E4, C-HEGA®-11, n-Octyl-β-D-glucoside, FOS-Choline®-12, FOS-Choline®-8 fluorinated, CHAPS, CHAPSO, ANAPOE®-58, CYMAL®-4, Sulfobetaine 3–10 and LDAO) HRV 3C protease activity was reduced to ~ 50% relative to the control reaction and the activity decreased further, below 50%, relative to the cleavage in the control reaction in the presence of another twelve detergents (MEGA-9, HEGA®-9, n-Hexyl-β-D-glucopyranoside, ZWITTERGENT® 3–12, MEGA-10, Pluronic® F-68, HECAMEG®, CYMAL®-3, NDSB-256, FOS-MEA®-10, LysoFos™ Choline 12, and Sodium dodecanoylsarcosine). HRV 3C protease activity was completely abolished in the presence of eight detergents (C-HEGA®-10, C-HEGA®-9, HEGA®-8, CYMAL®-2, n-Decyl-N,N-dimethylglycine, MEGA-8, FOS-Choline®-8 and ANAPOE®-20). In 2011, Vergis and Weiner [4] reported a study on the cleavage activity of HRV 3C protease in the presence of a wide range of detergents at 25°C. Although the substrates are different and half of the detergents tested are different, comparison of both datasets for the same detergents show that, in most cases, HRV 3C protease activity is preserved better at 4°C. For example, whereas Vergis and Weiner [4] report little or no cleavage in the presence of FOS-choline-12, HRV 3C protease cleaves over 50% of the target protein at 4°C. In contrast, cleavage is better at 25°C in the presence of C8E4, HECAMEG and n-undecyl-b-D-maltoside [4]. On the basis of structure of detergents, it has been observed that HRV 3C protease is inhibited to less extent by maltoside and glycol ether containing detergents.

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