The Fold And Assembly Of C-reactive Protein In Escherichia Coli

C-reactive protein(CRP)is an acute phase plasma protein.The plasma concentration of CRP can increase up to 1000-fold in response to tissue damage or infection,so it is a significant clinically protein.It is also associated with many human diseases,for example tissue damage,cardiovascular disease,atherosclerosis,etc.CRP consists of five noncovalently associated subunits arranged symmetrically around a central pore.The CRP subunit consists of 206 amino acids folded into two antiparallel ? sheets.Electron microscopic and crystallographic data indicate that in the assembled CRP pentamer all subunits have the same orientation.Thus,the molecule has two faces,a 'recognition' face exhibiting the five PCh-binding sites and an 'effector' face containing the Clq-binding sites.However,less is known about the mechanism of cellular folding and assembling process of CRP.First of all,we successfully obtained the secreted native C-reactive protein by co-expressing with kil gene and fusion with the signal peptide of alkaline phosphatase(ALP)of E.Coli and verified by ELISA with anti-CRP antibody.Then,we constructed various CRP mutants in the model of sALP-CRP kil and expressed in E.Coli.We found that aal-31 plays a guiding role in the fold of CRP,and then CRP form a stable core which lie in aa32-101.a helix consisted of aa 168-176 plays an important role of formation and stabilization of disulfide bonds of CRP.The cellular ratio of disulfide bonds come down dramaticlly and native CRP can't form without the integrate a helix.Disulfide bonds formation is an important process of CRP formation.The function of it is likely to lock the domains where the two Cys are located at and keep the domains stabilization.Combined with calcium is necessary for CRP formation.140D,61N and 150Q are essential amino acids for calcium ions combining.Ionic bonds of subunit is also necessary for CRP formation,especially 155D-118R which play a major role.With the mutation of D155A,CRP cann't normally form.Before subunits used to assemble,it has a preferred structural configuration.With above all,we can draw the fold and assembly of CRP in Escherichia coli into three stages.Firstly,CRP form a stable core.Secondly,aa101-168 and aa176-206 form its secondary structure and position.Lastly,five CRP subunits assemble into native CRP.