The expression of the HIV-1 protease in the laboratory is demanding due to its high cytotoxicity, which makes its expression in bacterial expression systems such as Escherichia coli difficult. To overcome these challenges, fusing HIV-1 protease with solubility-enhancing tags helps mitigate its cytotoxic effect and boost its expression as a soluble protein.
Therefore, this review focuses on the expression of the bioactive HIV-1 protease using solubility-enhancing fusion tags in Escherichia coli and summarizes the characteristics of the different common fusion tags that have been used in the expression of the HIV-1 protease. This review will help researchers to choose the protein fusion tag for HIV-1 protease expression.
SUMO proteases are a general class of enzymes that specifically remove post-translational protein modification (PTM) known as a ubiquitin-related small modifier (SUMO), which belongs to the PTM class of ubiquitin and/or ubiquitin-like proteins (UBL). The enzyme is commonly known as „SUMO protease” is the Ubl-specific protease 1 (Ulp1) from Saccharomyces cerevisiae.
This was the first of this class of enzymes to be isolated. The SUMO protease specifically cleaves the SUMO moiety in a „scarless” manner. The SUMO protease recognizes the tertiary structure of the ubiquitin-like SUMO domain and hydrolyzes the peptide bond in the x-Gly-Gly-x sequence after the Gly-Gly bond, at the C-terminal end of the SUMO domain. In addition to the cleavage of wild-type SUMO-modified proteins, SUMO protease is used to cleave recombinant SUMO fusion proteins.
The SUMO domain is a known solubility-enhancing fusion tag used in the expression of recombinant proteins. A histidine tag can also be added to the N-terminus of the SUMO domain as a purification tag. This SUMO protease product carries a 6x histidine tag. Therefore, it can be easily removed together with the cleaved SUMO domain, after the digestion reaction.
SUMO protease is active over a wide range of temperatures (2–30 ° C), ionic strengths (0–400 mM NaCl), and pH ranges (6–8.5). However, its activity may vary depending on the substrate and conditions. Researchers will need to optimize their specific reaction conditions. As an initial suggestion, 20 units of SUMO protease per mg of the target protein can be used for 1 hour at 30 ° C, or overnight at 2-8 ° C. Cleavage efficiency can then be estimated by SDS-PAGE. If necessary, the amount of SUMO protease can be adjusted. SUMO protease works best in the presence of reducing agents, eg 0.5-2 mM DTT. DTT in the reaction mix can significantly improve cleavage efficiency, especially during longer incubations.
Store the reconstituted product at –20 ° C. It is recommended to reconstitute the enzyme in 100 µL of water or 50% (v / v) glycerol, supplemented with 1 mM DTT. Water / DTT solutions should be stored in frozen aliquots to avoid freeze-thaw cycles, which can adversely affect protease activity.