A Preliminary Study On Structure And Function Of Rv3899c In Mycobacterium Tuberculosis

Tuberculosis(TB)is a chronic and delayed infection,which is caused by Mycobacterium tuberculosis.And there is a hypothetical protein in Mycobacterium tuberculosis,whose function are currently unknown,but this kind of proteins are real existence.This kind of hypothetical proteins in Mycobacterium tuberculosis account for more than half of total proteins.In this article,Rv3899c belongs to hypothetical Mycobacterium tuberculosis proteins,it is predicted that Rv3899c is highly conserved in Mycobacterium species.It was identified as a potential immunogen in the recent study.The gene encoding Rv3899c was amplified by polymerase chain reaction(PCR)using M.tuberculosis H37Rv genomic DNA as a template,and the PCR product was cloned into a pET-28a expression vector,then a pET28a-Rv3899c prokaryotic expression vector has been constucted.IPTG was added to the culture medium to induceprotein expression.Western blotting method indicated that the recombinant protein was expressed correctly.The recombinant protein was obtained after two purification steps comprising of nickel-affinity chromatography and size-exclusion chromatography,and yield high-purity recombination protein with His-tag.In order to resolve the structure of Rv3899c,initial screening was performed by Crystal Screening Kit.The crystals of Rv3899c appeared after about 7 days under the condition of Index78,Index83,Index84,Index95.Various optimization procedures such as altering protein concentration and the temperature of crystal growth were tried in order to obtain diffraction-quality crystals.N-terminal sequencing indicated that the protein fragment began with GATAG,indicating that the crystallized protein comprised residues 184-410 of Rv3899c,The crystals diffracted to a resolution of 1.90 A.Heavy-atom screening is currently underway in order to solve the three-dimensional structure by multiple isomorphous replacement.In order to explore the biological function of Rv3899c,both of the GST-Pull down and Mycobacterium tuberculosis protein chip technology were used to find proteins which ineract with Rv3899c.Through protein chip technology,we identified 84 proteins,including the stronger signal as Rv3420c,Rv3709c and Rv0914c.Their functions are to make the N-terminal alanine acetylation,make L-aspartate phosphorylation and degradation pathways involved in lipid metabolism.About the authenticity of their interactions also need other ways of in vitro or in vivo further validation.