Study On Expression And Function Of Genes Sso0660 And Sso0661 From Sulfolobus Solfataricus

Sulfolobus is a model organism for archaeal genetic,physiological and biochemical studies.Analysis of the S.solfataricus P2 genome sequence has revealed a protease inventory of at least 37 genes encoding putative proteases,protease subunits,or peptidases.However,to date,only six proteases or peptidases have been characterized including Sso2045,Sso2154 and CPSso etc.None of the remaining putative proteases have yet been analyzed.For example,Sso0660 and Sso0661,which were annotated as putative zinc-dependent proteases and TldD/E homologues in the S.solfataricus P2 genome database since they showed similarity to bacterial TldD/E.Genetic studies suggested that TldD and TldE of E.coli could have proteolytic activity and form a protease complex in vivo but their protease activity needs to be demonstrated biochemically.Furthermore,crystallographic analysis of Thermotoga maritima TldE(PmbA)failed to detect any coordinates for metal ions in the protein structure or any structural domain of hydrolase.Yet,there has not been any report on biochemical characterisation of a TldD/E homologue in the current literature.As a consequence,it remains to be demonstrated whether or not TldD/E encode proteases and further experiment will be needed to unravel the physiological role of these proteins.In this paper,we got some results as follows:1.Homologous and heterologous expression of Sso0660 and Sso0661 were carried out by constructing expression plasmids pSeSD-0660,pSeSD-0661,pET30a-0660 and pET30a-0661 etc.Heterologous recombinant proteins formed predominantly inclusion bodies while homologous recombinant proteins were present in the soluble fraction of the cell lysate.By gel filtration,mass spectrometry and point mutation,the molecular weight of Sso0660 monomer was confirmed as 49851 Da.Two Sso0660 monomers firstly formed a dimer by intermolecular disulfide bond between C416 and then formed 36-polymer on this basis.Homologous recombinant Sso0661 was identified as a monomer with molecular weight of 47346 Da.2.For the first time it has been demonstrated that Sso0660 and Sso0661 exhibited protease activities.The recombinant enzyme,which was purified from inclusion bodies after denaturation and refolding,was active in degrading FTC-BSA,azocasein and gelatin as same as the enzyme purified from the soluble fraction and their optimal reaction pH was 7.0.The optimal temperature of homologous and heterologous recombinant Sso0660 were found to be at 75 ? and 55 ?,respectively.The optimal temperature of recombinant Sso0661 was 65 ?.Protease inhibitor studies indicated that both Sso0660 and Sso0661 encoded metallproteases because EDTA and o-phenanthroline,two well-known metalloprotease inhibitors,either abolished completely or strongly inhibited the protease activity of the enzyme.One mole of zinc was found to be associated with each mole Sso0660 molecule whereas zinc was absent from the purified Sso0661 enzyme by flame atomic absorption.Sequence alignment results showed that TldD and TldE had the common conservative "DDEG" motif and only TldD had "HExxxH" motif and the cysteine residue at their C-terminal.Site-directed mutagenesis unraveled that "HExxxH" and "DDEG" motifs in Sso0660 sequence were involved in zinc binding.D278E changed the cleavage site,suggesting that it could play a role as catalytic center.3.Homologous overexpression of Sso0660 led to a possible link between Sso0660 and programmed cell death(PCD).This is because upon Sso0660 over-expression,the cells exhibited genomic DNA degradation,increased intracellular caspase-like activity and seriously growth arrest,highlighting the importance of archaeal TldD superfamily members in the regulation of programmed cell death(PCD)in archaea.Owing to be sensitive to generic caspase inhibitor Z-VAD-fmk,exhibiting hybridization signals with anti-human caspase-8 antibody and showing caspase-8-like activity,Sso0660 was deduced to be caspase-8-like and death protease and play important role in archaeal PCD.This work constitutes the first report of identification of a caspase-like protein in archaeal domain.4.Co-expression vector pSeSD-0660/0661 was constructed and Sso0660/0661 complex was found by co-purification and co-immunoprecipitation.We presumed that TldD and TldE might restrict each other in vivo,however,tangible interaction mechanisms remain to be reveal in the following work.