A Preliminary Study Of The Function And Crystallization Of The Recombinant Dnaj From Streptococcus Pneumoniae

Background Streptococcus pneumoniae is a conditional pathogenicbacterium, which has become one of the important pathogens ofcommunity infection. SpDnaj is heat shock protein40of Streptococcuspneumoniae, which plays an important role in the process of proteinfolding and transporting. The mechanism of SpDnaj recongonizing thepeptide (substrate) could provide key information to look for a newantibacterial drug. To date, three crystal structures of Dnaj have beenreported, including Ydj1from yeast, DnaJ from Klebsiella pneumoniae andCaenorhabditis elegans, whereas there is no report on the structure offull-length DnaJ and the mechanisms are still unclear.Objective Consequently, to determine the three-dimensional structure ofSpDnaj and the mechanisms of binding peptide, we have initiated thedetermination of its three-dimensional structure by X-ray crystallographyand determined its ability to recongonize peptide.Method The gene encoding SpDnaJ was amplified from genomic DNAof Streptococcus pneumoniae and inserted into PET28a to construct theplasmid PET28a-SpDnaj. The SpDnaj protein was purified by metal ion affinity chromatography column (Ni-NAT) and anion exchangechromatography column (DEAE Sepharose Fast Flow). The aggregation ofSpDnaj in the solution was analyzed by sieve chromatography (Superdex200). The crystals of SpDnaj and Se-SpDnaj are grown with bothsitting-drop and hanging-drop vapour-diffusion methods, and crystaldiffraction data by X-ray diffractometer were collected on beamlineBL17U1of the Shanghai Synchrotron Radiation Facility (SSRF). ITC(Isothermal Titration Calorimetry) and SPR (Surface Plasmon Resonance)technology was used to determine the capacity to bind to substrate.Result It is succeeded in constructing recombinant plasmidPET28a-Dnaj, expressing and purifying SpDnaj protein with the purity of85%. Gel filtration experiments suggest that the protein is arranged as atetramer in the solution. X-ray diffraction datas, including SpDnaj,Se-SpDanj, SpDnaj-P5-10, were collected from a single crystal onbeamline U17O at SSRF. The SpDnaJ crystal belonged to the space groupI222or I212121, with unit-cell parameters a=47.68, b=104.45and c=234.57. Based on the molecular weight of SpDnaJ (about40.5kDa) andspace group, solvent-content analysis indicated that one molecule could beaccommodated per asymmetric unit, suggesting a VMvalue of3.243Da-1and a solvent content of62.08%. However, molecular replacement andmultiple-wavelength anomalous dispersion methods failed to analyze thestructure. ITC and SPR results showed that SpDnaj combined with peptide P5-10.