The Expression, Purification And Function Of Different Grn Fragments Of PGRN And Its Derivative Protein Atsttrin

PGRN is a multifunctional protein involved in many important physiological processes,but its mechanism is unclear.Therefore,many worldwide scientists are making great effort in this research area.Our laboratory PGRN has been carrying out the project of the molecular mechanism of its biofuntion.The mature PGRN can be proteolytically cleaved to generate Grn domains,which have important biological functions.Recent study indicated that the regulation of Gm D domain and Gm E domain are related to glioma proliferation and survival of motor neurons,respectively.Grn B stimulates the proliferation of murine keratinocytes;hGrn A demonstrated potent inhibition effects on the growth of breast cancer cell line.Recent study on mice model indicated that PGRN plays a similar role as Atsttrin in the pathogenesis of inflammatory arthritis.Atsttrin is an engineering protein,composed of half units of granulins A,C and F plus linkers.However,the molecular mechanism of PGRN in anti-inflammation is still not clear.In addition,the PGRN relevant peptides need to be further study.In order to study the molecular mechanism of Grns and Atsttrin,we use different expression system executing the protein expression.Then,we preliminarily explore the functions of Grns and Atsttrin.The research include following parts:1.Constructing the yeast expression vector:PichiaPinkTM Exession System andpPIC9K in GS115 expression systems are currently utilized at our laboratory,which are developed from yeast pichia pastoris by using different screening theory and method.So far,there is no available data regarding the comparison of these two systems.We got the gene of a-factor and HSA by PCR,then introduced the secretion signal peptide?HSA gene?6his tag and multiple cloning restriction sites to the initial commercial vector pPink-HC and pPIC9K,getting four universal expression vector named pPink-HC-a-factor-MCS-6his?pPink-HC-a-factor-HSA-GGGGS-MCS-6his pPIC9K-MCS-6his?and pPIC9K-HSA-GGGGS-MCS-6his successfully.After that,we aimed to comparing these two expression systems,simultaneously evaluated the expression level between expressing Grns single and expressing it fused with HSA.2.Exploring the regulation of protein expressing of Grn E:Firstly,we got the 8 Grns fragment by PCR,and then insert the Grn E sequence into the four universal expression vectors(pPink-HC-a-factor-Gm E-6his?pPIC9K-Gm E-6his?pPink-HC-a-factor-HSA-Grn E-6his and pPIC9K-HSA-Gm E-6his)by enzyme digestion and ligation.These vectors are linearized by the appropriate restriction enzymes,and then transformed into pichia pink strain 1 and GS115 by electroporation.After screening expressing recombinant strains in selecting plate,the stains induced to expressing target protein by methanol.We detected the yeast expression supernatant by SDS-PAGE and Western blot.The result showed that Grn E can express in both expression system,and their expression effect is consistent.Comparing the pPIC9K in GS115,the PichiaPinkTM system is more easy to operate?Saving time,and recombinant screening is more intuitive and does not require expensive antibiotic selection.3.Processing the expression and purification of Grns and HSA-Grns:The other Grns(GrnP?GrnG?GrnF?GrnB?Grn A?Grn?Grn)were constructed into pPink-HC-a-factor-MCS-6his and pPink-HC-a-factor-HSA-MCS-6his expression vector,expressing the protein Grns and HSA-Grns as same step with Gm E.The results of SDS-PAGE and Western blot showed Grns fusion with HSA expression promoted the protein expression and stability.As Grns could be degraded,the protein expression of Grns is undetectable.The protein concentration of Ni-NTA purified HSA-Gms in supernatant was up to 0.5 mg/mL,which presented as a clear and high specific band by SDS-PAGE.4.Study on the function of Grns:The purified proteins(HSA-6his?HSA-Grn P-6his?HSA-Grn G-6his?HSA-Grn F-6his?HSA-Grn C-6his?HSA-Gm D-6his?HSA-Grn E-6his?HSA-Grn B-6his?HSA-Grn A-6his)were added into PC 12 cell line with a concentration of 10 ng/mL.HSA-6his was utilized as a negative control.The cellular growth conditions of axons were daily monitored and recorded using an inverted microscope.The observed results showed that the treatment of HSA-Grns HSA no effect on the growth condition of axons.5.Expressing and purifying the PGRN derived protein atsttrin,then study its function in dermatitis mouse model:To get the pRSET-B/atsttrin vector,the atsttrin gene sequence was generated by overlap PCR,and then cloned into the prokaryotic expression vector.The expression vector was electro-transformated into E.coli BL21(DE3),which was able to normally express the protein atsttrin.In order to understand its anti-inflammatory activity and mechanism,we established the dermatitis mouse model,which was treated with purified atsttrin by intraperitoneal injection.The ear thickness and inflammatory factors related dermatitis were measured in both treated group and control group.The result showed that the ear thickness of atsttrin treated dermatitis mouse were reduced dramatically and the protein expression level of inflammatory factors IL-1??IL-6?Ptgs2 were decreased.In summary,four common yeast expression vectors were constructed and Gm E and HSA-Gm E were expressed in two yeast expression systems,respectively.The result showed that Grn E was able to normally express in the two systems,and the PichiaPinkTM system presented a better expression timeliness and operational aspects.To our best knowledge,this study is the first report of the evaluation of the two yeast expression systems,which could provide useful guideline for the further application.In following study,the other seven Grns and HSA-Grns were expressed in PichiaPinkTM system.The results indicated that HSA-Grns presented higher protein expression level and better protein stability.Eight HSA-Grns were highly expressed and HSA-Grns-6his was purified by Ni-NTA.Our finding could be useful guideline for antibody preparation.To explore the bioactivity and function of Grns,we studied the effect of Grns on cellular axon growth condition in PC 12 cells,which were treated with HSA-Grns-6his and HSA-6his.The result showed that cells treated HSA-Grns were similar with negative control.On the other hand,we successfully constructed pRSET-B/atsttrin expression vector and purified expressed 6his-atsttrin.Then,the mechanism was explored in dermatitis mouse model.In atsttrin injected mice model,the ear thickness was significantly reduced and inflammatory cytokines IL-1?,IL-6,Ptgs2 expression level was significantly lower,which suggested that atsttrin HSA potential anti-inflammatory effect.