| The diabetic can emerg questions that insulin can not play a normal role in target cells or insufficient supply of insulin,Leading to conversion disorder of sugar,fat and protein.Moreover,metabolism disorder about water and electrolyte has become systemic disease.Diabetes mellitus includes type 1(insulin-dependent)and type 2(non insulin dependent,which more than 90%patients with type 2 diabetes mellitus.The hepatokine fibroblast growth factor 21(FGF21)is known as a key metabolic regulator,increases insulin sensitivityand causes weight loss.Kinds of clinical studies have demonstrated that FGF21 serves as a potential therapeutical target for the treatment of obesity and type 2 diabetes.The expression of recombinant proteins in Escherichia coli is one of the most common host.Extracellular expression of recombinant protein have several advantages,such as promoting the correct refolding of protein in Escherichia coli,effectively reducing the formation of inclusion body,simplifing the purification process and other advantages.Besides,Escherichia coli has clear genetic background,simple operation,short growth cycle,low cost,easy to large-scale cultivation.The traditional method of using Escherichia coli expresses heterologous protein,there are some problems including lack solubility of protein,unfolding and refolding of inclusion body.That result shigh production costsand time consuming.So expect through the efficient construction of Escherichia coli secretion system to increasing FGF21 secretion.Due to Escherichia coli Type? secretion system is not through the periplasmic space,it can effectively reduce the protein secretionconsumingin the process.Typel components mainly including three parts:HlyA upstreamsimilar signal peptide,HlyB-HlyD and TolC.Through the construction of Escherichia coliType?secretion system to secrete FGF21 protein expression.However,the analysis of the secretion of FGF21 isn't obvious.So,expecting to construct E.coil Type?secretory system in order to increase FGF21 secretion.Construction of Type II secretion system in E.coli included signal peptide screening,molecular chaperone co-overexpression and protease knockout.FGF21 gene was cloned into pET-19d vector,fidentify suitable host cells.BL21 and its derivative strains are widely used in expressing recombinant protein,because those strains are all protease deficient strains(e.g.OmpT and Lon protease)and they can effectively avoid the recombinant protein degradation.OmpT protease specifilycut T7 RNA polymerase,Lon protease can degrade heterologous recombinant proteins fastly.The experiment choses BL21(DE3)as host strains to secrete FGF21;Second,Seven plasmids including different signal peptide(malE,ompF,phoA,ompA,lamB,DsbA and TorT)were constructed,and be transformed into BL21(DE3).Shake flask fermentation were compared and the results showed that ompA signal peptide was more suitable for secretion of FGF21.Third,molecular chaperones plasmids were constructed and co-overexpression with pET-19d-SPompA-FGF21 to increase the solubility of protein.The results showed that DsbC overexpression can promote the secretion of FGF21.On account of recombinant protein can be degraded easily by some intracellular protease,Keio collection strain library was used to detect the effect of protease on secretory of FGF21.Proteases including Lon,ClpAP,ClpXP,HslUV,FtsH,OmpT,Prc,DegP,PtrA knockout strains fermentation were compared and it seems that lon,ptrA and HslV pknockout strains have positive effect on expression and secretion of FGF21.Two genes PtrA and HslV were successfully knockout by using CRISPR/Cas9 gene editing technology.However,the protease deficiency strains grew slowly and the secretion of FGF21 did not have a favorable effect.Through optimization and combination of effective factors,pET-19d-SPompA-FGF21 co-overexpression with DsbC by shake flask fermentation,the final secretion of FGF21 can reach 0.3mg/ml. |
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