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Cloning, Expression and Purification of DegQ Protease, a Bacterial Analog to Pregnancy Related Serine Protease (HtrA3)

The High Temperature Requirement A (HtrA) family of enzymes are a diverse group of serine proteases, present in both prokarya and eukarya, and are generally responsible for the degradation of misfolded proteins. The dysregulation of HtrA3 in human somatic cells gives rise to various forms of cancer and preeclampsia. DegQ is a bacterial analog to HtrA3 found in the periplasm of E. coli and has the properties of an HtrA protease: it is a serine endopeptidase that works to protect the cell during high temperature by degrading specific damaged proteins. Currently, the interactions that dictate which specific substrates DegQ degrades are unknown. Insights into this interaction could lay the foundation for studying the interactions of the human HtrA3 protease. Previously, four N‐terminal truncated versions of DegQ were identified in prokarya, indicating an N‐terminal signaling sequence. Substrate cleavage in each of these variants has not been characterized, therefore, the goal of this study is to select for the active version to further characterize. Initial experiments utilized molecular cloning, protein expression and purification to obtain each variant. Affinity tags, such as the histidine‐tag, has greatly simplified the purification of recombinant proteins. However, the presence of such tags can interfere with a protein's biological function. To circumvent this problem, experiments were designed to obtain the protein without the use of a tag. To this end, four PCR forward primers were designed for the appropriate coding region using E. coli as template genomic DNA, each with an NdeI site engineered at the 5′ end. The reverse primer for each was the same, and designed with a stop codon followed by a SacI site at the 3′ end. The PCR product was then ligated into a linearized dephosphorylated pGEM vector using TA cloning. Orientation of the gene was determined through the use of restriction enzyme cuts. This vector and the pET24b expression vector were digested with NdeI and the gene was ligated into the pET24b vector. Orientation was again tested using SacI sites, and analyzed by gel electrophoresis. Based upon the theoretical mass distribution of the cleaved oligonucleotides from the gel electrophoresis, plasmids with positive results were purified for verification through sanger sequencing. Each pET24b‐DegQ vector was individually transformed into BL21(DE3) E. coli cells, grown in LB broth and expressed with 0.1 mM IPTG for 4 hours at room temperature. Protein was purified using a DEAE‐sephacel column, followed by an ammonium sulfate precipitation, and further purification on a G‐75‐sephadex size exclusion gel filtration column. Ultimately the proteases were purified to homogeneity without the use of an affinity tag for comparison of proteolytic activity.