Construction Of The Eukaryotic Expression System Of Human Decorin And Its Activity Evaluation

Decorin(DCN)belongs to extracellular matrix small leucine-rich proteoglycan molecules family and was first isolated in 1978 by Professor Fisher from National Institutes of Health.The molecular weight of DCN is 39KDa and has been showed with a variety of significant biological activities.DCN presents the commendable inhibition on the occurrence and progression of tumors through the interactions with extracellular matrix proteins,cytokines and cell surface receptor.Previous studies found that loss expression of DCN would promote tumorigenesis.Meanwhile,it has been suggested that DCN has a TGF-p binding domain,the combination of DCN and TGF-p prevents TGF-p from combining to the other receptor,and then inhibits the biological activity and structural stability of TGF-?.DCN is also a new type of EGFR ligands,which could cause EGFR tyrosine kinase sustained inactivation,proceed to reduce EGFR/ErbB family receptor tyrosine kinase expression,then increase intracellular calcium anion level and p21,p27 genes expression,eventually act on suppressing tumor growth.Some researches show that,DCN is a novel antagonistic ligand of the Met receptor.The combination of DCN and Met receptor could lead to Met receptor expression and subsequently ubiquitylation and degradation,then cause significant degradation of?-catenin,which presents to be a Met receptor downstream effector.Recelt studies carried out that DCN could induce various reactions on EGFR to activate phosphotyrosine of EGFR autophosphorylation acid region selectively to produce different signaling cascade from EGF and cause degradation of EGFR to inhibit the activity of ErbB2 and ErbB4 receptor,ultimately contribute to tumor proliferation inhibition.It has also been mentioned that DCN could induce endothelial cell sprouting and activate intracellular pathways to affect tumor angiogenesis.Moreover,DCN shows the contribution to the down regulation of?-catenin,which was combined to E-cadherin in vitro,in vivo and xenograft models conditions.All these results attest the important role of DCN in tumor metastasis.On the basis of previous studies,we set up the eukaryotic cells(Pichia)protein expression system by genetic engineering techniques.The DCN protein from the expression system was purified by Sephadex G-75.The anti-tumor activity of DCN expression protein was tested by wound healing and MTT assay.The molecular mechanism of potential tumor inhibition activities by DCN would be explored by the followed Western Blot assay.1,Vector construction:Decorin sequence was obtained by PCR and then ligated to eukaryotic vector pPICZ?A.pPICZ?A-decorin was transformed into E.coli DH5a for amplification.Confirmed by enzyme digestion and sequencing.pPICZaA-decorin was linearized and transformed into Pichia Pastoris strain GS115 by electroporation to complete DCN-yeast expression vector.2.Expression and purification of recombinant protein:a large number of recombinant plasmids were extracted and linearized to integration with Pichia gene by electroporation.The transformed vector yeast was cultured on YPD solid medium with bleomycin(Zeocin)and sorbitol at 30°C for 3 to 5 days.The single colony was inoculated onto YPD solid medium containing high concentrations of bleomycin(Zeocin)at 30? for 3 to 5 days.Well-conditioned single colony was inoculated in a 20ml YPD liquid medium then was cultured on a shaker overnight at 37?.3 ml of the former medium was seeded in new 100ml YPD liquid medium at 37? on a shaker until OD600?1.0,then 0.5%methanol was added to induce recombinant protein expression.The additional methanol was added per 24 h later,the medium was collected after 4 days.The supernatant was collected by centrifugation for 10000 rpm/min,15min at 4?.Sephadex G-75 was then used for filtration to get the crude protein.After eluted,concentrated,desalted and lyophilized,the recombinant DCN protein was obtain.3.Biological activity analysis:cell wound healing and MTT assay was used to determine the cell growth inhibition activity of the recombinant DCN protein.The results reveal that the recombinant DCN protein shows expected growth inhibition on HCT-116 colon cancer cell line.4.Molecular mechanism study:Western Blot assay was used to detect protein expression changes of CDK4,Cyclin D1,?-catenin and E-cadherin.The Results show that the protein expression of CDK4,Cyclin D1 and ?-catenin was down-regulated and E-cadherin was up-regulated.In conclusion,we established Pichia expression system for DCN gene expression with correctly folding and processing modification after translation recombined vector to get recombinant DCN protein product,which was turned to be closer to the natural structure.The results show that the recombinant DCN protein has effective activities on inhibiting the growth and metastasis of colon cancer cells,which supports the application of DCN as a therapy agent for colorectal cancer treatment.