1.High-level Prokaryotic Expression And Purification Of Death Domain Superfamily With MBP Tag 2.Structural Basis Of NLRP1 And ASC Inflammasome

1.High-level Prokaryotic Expression and Purification of Death Domain Superfamily with MBP TagDeath domain superfamily are compact structural motifs that play critical roles in many biological processes including inflammation and apoptosis.Due to mediating protein-protein interactions,these death domains are notoriously difficult to deal with when they overexpressed in vitro.In this study,we found that the maltose binding protein(MBP)is very effective in improving the behavior of many death domain superfamily members as a fusion protein.In order to achieve high-level expression of death domain superfamily in E.coli,we designed two MBP-tagged expression vectors based on a pET30a backbone:One with a short flexible noncleavable linker,the other with a Tobacco etch virus(TEV)cleavage site.Soluble protein was purified from cell lysate by HisTrapTM IMAC column and Superdex-200 gel filtration column.We tested seven targets of death domain superfamily,including two PYDs、three CARDs and two DDs,which are all challenging to obtain without a tag.We expressed and purified these death domain proteins successfully as soluble and pure protein with both vectors.Among them,three proteins were crystallized successfully.My study demonstrated that both recombinant MBP expression vectors significantly enhance the yield and solubility of fusion proteins and can help the target protein crystallize.2.Structural Basis of NLRP1 and ASC InflammasomeInnate immune system includes several pattern recognition receptor families that initiate immune responses during microbial infections through a variety of mechanisms.NOD-like receptor(NLR)is a class of pattern recognition receptors expressed in the cytoplasm,which is composed of about 20 members in the human genome.NLRP1 is the first discovered member of the NOD-like receptor(NLR)family and is also known as CARD7,CIDED,DEFCAP and SLEV1.NLRP1 is mainly composed of five domains,namely NACHT,NAD,LRR,FIUND and CARD,while human NLRP1(hNLRP1)has one extra N-terminal PYD domain compared to mouse NLRP1(mNLRP1).When the organism is infected by external pathogens,activated NLRP1 recruits downstream ASC and Caspase-1 through the CARD domain to form NLRP1 inflammasome,which mediates cell death,maturation of IL-1β,IL-18 and cause inflammation.,At present,the specific molecular mechanism of NLRP1 inflammasome activation is still unknown.Therefore,this thesis aim to explore the molecular mechanism of hNLRP1 inflammasome activation and its biological function in vivo by investigating the interaction between hNLRP1 and hASC.By constructing a recombinant expression vector harboring the maltose-binding protein(MBP),the corresponding fusion proteins were expressed and purified.I found that hNLRP1-CARD domain interacts with its self in solution and in vitro in yeast,the homotypic interface is confirmed by mutagenesis.In addition,yeast two-hybrid and mutagenesis experiments showed that the hNLRP1-CARD dimeric interface may be necessary for interaction with hASC-CARD.Lastly,a crystal structure of hASC-CARD at a resolution of 2.8 A was obtained,which conforms to the classical CARD structure.My thesis study provided novel insights for the molecular of NLRP1 inflammasome activation.