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.
Drug Discovery and Structural Biology group, Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan.|Pakistan Institute of Engineering and Applied Sciences, P.O. Nilore, Islamabad, Pakistan.
Raheem
R
Ullah
Drug Discovery and Structural Biology group, Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan.|Pakistan Institute of Engineering and Applied Sciences, P.O. Nilore, Islamabad, Pakistan.
Majid Ali
MA
Shah
Drug Discovery and Structural Biology group, Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan.
Soban
S
Tufail
Drug Discovery and Structural Biology group, Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan.
Fouzia
F
Ismat
Drug Discovery and Structural Biology group, Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan.
Muhammad
M
Imran
Drug Discovery and Structural Biology group, Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan.|Pakistan Institute of Engineering and Applied Sciences, P.O. Nilore, Islamabad, Pakistan.
Mazhar
M
Iqbal
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
Osman
O
Mirza
Drug Discovery and Structural Biology group, Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan.|Pakistan Institute of Engineering and Applied Sciences, P.O. Nilore, Islamabad, Pakistan.
Moazur
M
Rhaman
D000086042:3C Viral Proteases; D002021:Buffers; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D003902:Detergents / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012229:Rhinovirus / Q000378:metabolism*; D012995:Solubility; D012996:Solutions / Q000378:metabolism*; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism*
D002021:Buffers; D003902:Detergents; D011993:Recombinant Fusion Proteins; D012996:Solutions; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases