Prkaryotic Expression And Purification Of NEK7 Kinase And Preliminary Screening Of Inhibitors

NIMA（never in mitosis,gene A）serine/threonine kinase 7（NEK7）is a regulator of mitosis spindle in mammals and considered as a drug target of inflammasome related inflammatory diseases.However,most commercially available or reported recombinant NEK7 proteins are either inactive,or low purity,or fused with bulky tags（needed to be removed further）,or in insect expression system（high cost and time consuming）.The very low purity or high cost limit the pharmacological studies and development of NEK7 inhibitors.As what caused the low purity of NEK7 in prokaryotic cells was unknown,it is necessary to seek the reason before solve it.Otherwise,it will be difficult to create a simple,rapid and time-saving method to express and purify highly pure and active NEK7,especially with a minimal tag in E.coli.The recombinant human NEK7 protein of the target protein was expressed and purified in large quantities in prokaryotes,and the obtained recombinant human NEK7 protein was used for subsequent studies,laying a foundation for finding natural small molecule compounds that can interact with NEK7.NEK7 protein expression vectors with N-terminal His-tag and C-terminal His-tag were constructed,and the expression of E.coli BL21（DE3）or BL21（DE3）pLysS were induced.Soluble NEK7 protein were purified by Co2+ affinity chromatography column or Ni2+ affinity chromatography column.Then NEK7 protein with higher purity was obtained by Capto S exchange chromatography column and Sephacryl S-100 chromatography column.When high purity NEK7 protein was obtained,to ensure whether the NEK7 protein was consistent with the theoretical one,a MALDI-TOF MS/MS spectrometry analysis was applied.Whether the Kinase activity was retained in the purification was detected by the kinase-Glo Assay,and whether the Biophysical property of NEK7 Kinase was correct was detected by the Thermal Shifting Assay,and this method could be used to screen Nek7 ligand compounds.Here,we report a comparative study of NEK7 full-length expression,N-terminal His-tag and C-terminal His-tag,and elucidate the reason for the low purity of NEK7 in E.coli.Our results demonstrated that N-terminal tagged protein was toxic to E.coli,resulting in incomplete translated products.The C-terminal tagged NEK7-His6 was higher pure than that of an N-terminal tag.The Ni2+ resin one-step purification led to a purity of 91.7%,meeting the criteria of most kinase assays.With two-step and three-step procedures,the protein purities were 94.7%and～100%,respectively.The NEK7 purified in this work maintained its kinase activity and correct conformation,as well as the ability of the compound to interact with proteins.And two small molecules with ligands that interact with NEK7 were screened through Thermal Shift Assay.In conclusion,our optimized protocol could produce good purity and yield of His tagged NEK7 in E.coli,and the kinase activity and biophysical characteristics of which are remained.The Thermal Shift Assay can be used for high-throughput screening of NEK7 ligand compounds.Secondary metabolic re-searches on the genus of Toona have led to the identification of a lot of structurally-unique limonoids with various kinds of pharmacological activities,such as cytotoxic,anti-inflammatory,and anti-multidrug resistance（AMDR）activities.Through the extraction and separation of the effective components in Toona Ciliata M.Roem,we conducted the preliminary screening of the anti-inflammatory activity and Thermal Shift Assay for limonoids we separated.Ciliatone D is not a ligand compound of NEK7 by Thermal Shift analysis.Nonetheless,ciliatone D gave a potential inhibiting effect on NLRP3 inflammasome with IC50 values 9.7 ± 3.7 μM and 7.8 ± 4.9 μM against lactate dehydrogenase（LDH）and interleukin 1β（IL-1β）,respectively.Western Blot analyses of caspase-1,IL-1β,and gasdermin D（GSDMD）exhibited that 4 could act on nucleotide-binding oligomerization domain,leucine-rich repeat and pyrin domaincontaining 3（NLRP3）inflammasome and blocked cell pyroptosis.This research could provide support for the medical application of T.ciliate as natural inhibitors of NLRP3 inflammasome.