Global sales of biopharmaceutical products are expected to expand at an average annual rate of 12% between 2024 and 2034 to reach >1 billion USD. The biopharmaceutical products must be more than 99 % pure in order to meet regulatory standards. Biopharmaceutical products like recombinant proteins require multiple chromatographic steps like ion-exchange chromatography, hydrophobic interaction chromatography, reverse-phase chromatography, and size-exclusion chromatography to get ~99 % purity. Because of the multiple steps, the cost of the reagents increases, which raises the cost of production. Protein purification cost is a major contributor to the production cost. As a solution, including an affinity tag (His-tag, GST-tag) lowers the purification cost and the number of chromatographic stages. A protease, such as tobacco etch virus (TEV) protease, must be used to eliminate the affinity tags, though. The affinity tag removal procedure has been done in batches. Fresh protease is therefore needed for every batch. Thus, the cost of protease accounts for a significant portion of the cost of raw materials. This problem could be resolved by immobilizing protease, which would enable its reuse and lower the cost of raw materials.
Methods
In this study, the nucleotide sequence of wild type TEV protease gene (Genbank id 734212.1) was extracted from NCBI database and optimized for expression in Escherichia coli. To aid in purification, a 6x-histidine tag was added to the N-terminal end of TEV protease. A 10x-aspartic acid tag was attached to the C-terminal end. The aspartic acid tag would be negatively charged at pH 7.4 and would anchor the TEV protease to the anion exchange membrane.
The modified TEV protease gene was cloned into T7 Express lysY competent E. coli for TEV protease production. TEV protease was purified using a Ni2+-nitrilotriacetic acid column (His-trap) attached to an AKTA Go fast protein liquid chromatograph (FPLC). The enzyme assay was done using 5-carboxyflourescin (a florescent substrate). All chromatography and protease cleavage steps were s carried out at 4℃.
A commercial anion exchange hollow fiber membrane (Sartobind Q) was selected for immobilization of TEV protease. The column was washed with 20 mM Tris-Cl buffer at pH 7.4 and supplemented with 1 mM dithiothreitol (DTT). TEV protease requires a reducing environment to function; addition of DTT helps maintain a reducing environment. In order to determine how much TEV protease would be needed to saturate the column, the dynamic binding capacity of Sartobind Q was determined. Here, pure TEV protease was allowed to flow through the Sartobind Q column, and the flowthrough was monitored for presence of TEV protease. Once TEV protease was found in the flowthrough, further application of TEV protease to the Sartobind Q column was stopped.
Parallelly, a His-tagged human annexin A5 protein was expressed in E. coli and purified using a His-trap chromatography column using an AKTA GO FPLC system. Here a TEV protease recognition sequence was added in between the His-tag and the annexin A5. To this pure solution of annexin A5 protein, 1 mM of DTT and 100 mM of sodium chloride were added. This solution was passed through the TEV protease column at a linear flow-rate of 0.4 cm/h.
Once the annexin A5 solution was passed through the Sartobind Q column, the solution was passed through a His-trap column attached to an AKTA Go. The fraction of annexin A5 without the tag was present in the flowthrough. Elution was performed with 500 mM imidazole to remove the rest of the annexin A5 containing the His-tag. The proteins were analyzed using SDS-PAGE.
Results
As a result of optimization, the nucleotide sequence of the optimized TEV protease gene was improved for expression in E. coli. When compared to the native TEV protease gene, 58% of the nucleotides in the optimized gene were changed to match the codon preferences of E. coli. Post transformation in E. coli, the recombinant cells were cultured in 1 L of Terrific Broth. From 1 liter of culture, 23 mg of TEVp was purified using the His-trap chromatography column. The specific activity was found to be 1.17 U/mg. The Michaelis-Menten constant (Km) and catalytic efficiency (kcat/Km) were determined to be 0.63 μM and 0.9 μM-1min-1, respectively. In a study of this enzyme reported in the literature, kcat/Km was found to be 0.9 μM-1min-1.
The dynamic binding capacity curve was constructed by passing pure TEV protease through the Sartobind Q column. It was found that 3 mg of TEV protease was enough to saturate the anion exchange ligands (quaternary ammonium compound) in the Sartobind column.
Purified His-tagged annexin A5 (17mg) was passed through the column of immobilized TEV protease. The flowthrough from the Sartobind Q column was passed through a His-trap column, and 14 mg of annexin A5 was obtained in the flowthrough of His-trap column, while 3 mg was obtained in the eluate from His-trap column. The absence of a His-tag will not allow a protein to bind to Ni2+-NTA resin in a His-trap column, and the protein will come out in the flowthrough. Therefore, 82% of the His-tag on annexin A5 was removed by the protease.
Conclusion
According to existing literature, immobilized TEV protease systems have been developed and used to cleave affinity tags. However, in the existing studies, immobilized TEV protease has been used in batch mode. Also, the reaction time for each batch ranged between 10-24 hours. In this study, an in-line/continuous system with immobilized TEV protease has been developed for cleavage of affinity tags. In addition to facilitating reuse of the protease, the continuous affinity-tag cleavage system has the advantage that it requires >90% less time than the conventional batch procedure.