Expression, Protein Purification, Polyclonal Antibody Preparation Of Allantoicase(allC) From Dictyostelium Discoideum

The social amoeba Dictyostelium discoideum which phagocytosis soil bacteria for food is a unicellular eukaryote.Under favorable condition,it multiplies as a unicellular organism.Upon starvation,a pathway involving aggregation,mound,slug, culmination stages induces the formation of a fruiting body consisting of a head of spores supported on a stalk of vacuolated cells.Spores await dispersal and germinate in favourable conditions for amoeboid growth.Dictyostelium discoideum is an excellent system in which to study cell mobility,cell-cell signal transduction,cell type differentiation,development and so on for its unicellular and multicellular life stages and rich life phenomena.The membrane glycoprotein gp150 encoded by Lag C,play a role in cell-type specification by heterophilic interactions.A strain in which lagC is disrupted, AK127,undergoes normal chemotaxis,but is arrested at the loose aggregate stage.So we inferred that gp150 play a key role during the whole development of dictyostelium.It may be in the signal transduction way of cell differentiation and apoptosis.However the concrete signal route has not been known.To study the influence of the cell adhesion molecule gp150 on the expression of genes needing for D.discoideum development,mRNA differential display was used to analyse the differences of gene expression between the wild strain KAx-3 cells and the mutant strain AK127 cells.We got a differential cDNA fragment,602bp,between KAx-3 cells and AK127 cells developed for 14h.This sequence had been submitted to the NCBI GenBank and the accession number is AY894718.We did homologous sequence alignment by The Basic Local Alignment Search Tool(BLAST),and we found the 98%base sequence of the differential cDNA feagment was the same as (aUantoicase)allC,E-value is 0.So we concluded that the aUC gene was differently expressed between the wild strain KAx-3 cells and the mutant strain AK127 cells.The sequence of Dictyostelium discoideum was completed in 2005.The genome, about 34 Mb of DNA,consists of 6 chromosomes,mitochondrial DNA and ribosome DNA.allC is located in chromosome 3 coordinates 3183979 to 3185311.It.encodes a protein named allantoicase and its molecular weight is about 42kD.Allantoicase is an important enzyme in the degradation of purines.It catalyzes the conversion of allantoic acid into ureidoglycolate and urea.And the former was resolved into ammonia,CO2,glyoxylate.Ammonia is an important signal molecule which regulate the development of Dictyostelium.Ammonia inhibits stalk cell differentiation and promotes differentiation of pre-spore cells to spores by suppressing DIF cumulation.Thus we cloned the allC and got the polyclonal antibody of allC for studying the gene function in Dictyostelium development.Total RNA was isolated from KAx-3 cells which had developed for 14h.After reverse transcription we got the first strand cDNA.Then we cloned the allC by PCR. The allantoicase gene(allC)of Dictyostelium discoideum was cloned into the fusion expression vector pET-32a and expressed in E.coli BL21(DE3)host cells.After the expression strain was induced for 4 hours by 1mmol/L IPTG,the fusion protein allantoicase was successly expressed and detected in inclusion bodies.The fusion allantoicase accounted 37%of total bacterial proteins.Inclusion bodies dissolved in the solusion of 8mol/L urea was purified by NI-NTA superflow cartridge under denaturing conditions.We got the fusion allantoicase with purity quotient of 70%.The inclusion body protein band in SDS-PAGE was excised and the protein was extracted.Then the purified protein was injected into New Zealand rabbits to induce immunoreaction.The induction with four injections lasted for 45 days,and then the anti-serum was prepared.Western blotting showed that the anti-serum(diluted to 1: 2000)had specific antigen-antibody recognition to the fusion allantoicase.We got polyclonal antibody of allC which supplies good tool for researching the function of allC during dictyostelium development and for studying the interaction between allantoicase and gp150.