Expression,Purification And Primary Screening Of Conditions For Crystallization As Well As Antibody Production Of Strongyloides Stercoralis Protein Kinase RIOK-2

Protein kinase is a family of kinases that has the ability to regulate protein substrates’ activity and stability via phosphorylation, which can be divided into two subfamilies based on the difference of their structure one is eukaryotic protein kinase(ePK) and the other one is atypical protein kinase(aPK). The RIO group belongs to the aPK sub-family, and is consists of four highly conserved members, RIOK-1, RIOK-2, RIOK-3 and RIOK-B. Both RIOK-1 and RIOK-2 are important in organisms for their existence from archaea to humans. RIOK-1 and RIOK-2 have been observed to participate in ribosome biogenesis and cell cycle progression in yeast and human cells. No analogous combination of a wHTH domain and a kinase domain has been previously reported regarding RIOK-2, indicating that RIOK-2 may have multiple functions. Among RIO protein kinases, RIOK-2 have important regulatory roles in organisms, thus RIO protein kinases have been predicted as a potential target for clinical drug development.Protein crystallography is an important aspect for exploring proteins structure, focusing on solving 3D structure of protein molecules and their complexes by X-ray diffraction. Protein crystal structure has the vital significance for understanding the mechanism, structure foundation and function of proteins. The crystal structures of Archaeoglobus fulgidus RIOK-2(Af-RIOK-2) and Chaetomium thermophilum RIOK-2(Ct-RIOK-2) have been studied experimentally, but there is still lack of knowledge about the structure and substrates of the RIOK-2 in the multicellular organisms. For this reason, the following studies were conducted:(1) Bioinformatic analysis of Strongyloides stercoralis RIOK-2The amino acid sequence of Strongyloides stercoralis RIOK-2(Ss-RIOK-2) was aligned with Archaeoglobus fulgidus RIOK-2, Chaetomium thermophilum RIOK-2 and Homo sapiens RIOK-2, showing significant similarities between these four sequences, which are 15.8%, 34.1% and 42%, respectively. The N-terminal that contains the RIO domain is highly conserved while the C-terminal differs between species.(2) Expression and purification of Ss-RIOK-2 wild type, D228 A mutant type and truncated typeThe prokaryotic expression vectors of three types of Ss-RIOK-2 were constructed, providing the high-purity protein(8~10 mg/mL) successfully. There is a significant degradation during the purification of both wild type and D228 A mutant type Ss-RIOK-2, indicating that the degradation can’t be related to catalytic activity of this kinase, as the 228 th aspartate was previously determined to be the key amino acid which controls the catalytic activity. Truncated Ss-RIOK-2 contains 1~329 amino acids of wild type(including wHTH domain and RIO domain). It is known that truncation can improve the stability of Ss-RIOK-2, but we were not able to obtain truncated Ss-RIOK-2 with a higher concentration, as the hydrophilic C-terminal was removed.(3) Screening of conditions for crystallization of wild type Ss-RIOK-2Initial screening was carried out by using the commercial crystal screening kits. After monitoring the crystallization conditions several times, we obtained the suspected crystal with a lamellar structure. When repeating this condition, along with optimizing, the precipitant and salt ion concentration, the same results were not reproducible, so it cannot be confirmed whether this lamellar structure is protein crystal or not.(4) Preparation of the polyclonal antibody of Ss-RIOK-2The purified recombinant proteins of Ss-RIOK-2 was used to produce polyclonal antibodies of anti-Ss-RIOK-2 in Japanese white rabbits. Western blot showed that the antibody was able to react with endogenous RIOK-2 in the total protein of Strongyloides stercoralis, indicating that this antibody can be used in the subsequent studies.In summary, recombinant plasmids of Ss-riok-2 wild type, D229 A mutant type and truncated type were constructed and expressed in E.coli, respectively. Then crystal screen of wild type Ss-RIOK-2 was performed. High quantity of anti-Ss-RIOK-2 antibody was also obtained in our study. All the results lay the foundation for further exploring the function of RIOK-2 in Strongyloides stercoralis and other studies, such as substrate screening of Ss-RIOK-2.