Construction And Optimization Of A RhFGF21 Secretory Expression System In B.subtilis

Fibroblast growth factor 21(FGF21)is known as a key metabolic regulator,increases insulin sensitivity,decreases blood TG level,reduces body fat and decreases body weight.Recent studies have shown that FGF21 serves as a potential therapeutical target for the treatment of obesity and type 2 diabetes.Bacillus subtilis(B.subtilis)is a gram-positive bacterium that has been approved by the FDA for human health(GRAS)organism,and is one of the common hosts for expressing recombinant proteins.Some proteins of B.subtilis could be secreted directly into the extracellular medium,the proteins are generally recognized as safe due to its lack of pathogenicity and deficiency in endotoxins.B.subtilis has the advantages of high-efficiency secretion and rapid growth.The expression of heterologous protein,which is derived from of eukaryotic cell is easy to cause the problems of protein soluble less and inclusion body much,resulting in high production cost and time consuming.The aim of this work is to explore a new method for improving the soluble expression and secretion level of Fibroblast growth factor 21(FGF21)in B.subtilis.In this study,we successfully expressed rh FGF21 in B.subtilis and subsequently designed a comprehensive expression strategy to increase souble expression and secretion of rh FGF21 through optimization at the levels of transcription,translation,protein folding,resistance to proteolysis,signal peptide cleavage processing,and translocation across the membrane in B.subtilis.1.We chose the B.subtilis 1A751 as the host strain,which is already deficient in two major extracellular proteases npr E and apr E.The plasmid p MATEF,an E.coli/B.subtilis shuttle vector which is derived from p MA5 F and uses the maltose-inducible promoter Pmal A instead of a constitutive promoter Phpa II,was used for expression the rh FGF21 protein.The results indicate that both expression plasmids(p MATEF and p MA5F)can successfully express rh FGF21 within cells.However,the expression level of rh FGF21 was still relatively low for large-scale purification,which constitutes a bottleneck.To overcome it,we attempted several strategies for the following experiments.2.We designed seven auxiliary mini-cistron cassettes,which can be used to reduce locally stabilized m RNA secondary structures and to enable efficient translation initiation,upstream of rh FGF21.These results indicated that cistron5(Gsi B)is an efficient mini-cistron cassette for enhancing the production of rh FGF21 in B.subtilis.3.The FGF21 protein contains one disulfide bond.In order to form correct disulfide bonds and to reduce the risk of protein misfolding,we constructed an overexpression vector with Dsb A and created a new recombinant strain named DSB.To futher assist in the formation of disulfide bonds,a gene trx A which hindered the formation of disulfide bonds in B.subtilis,was repressed named Trx F.The result indicated that the soluble expression level of rh FGF21 by the recombinant Trx F strain showed only a marginal increase.4.To further increase the production of rh FGF21 in B.subtilis,eleven molecular chaperones were overexpressed and integrated into the B.subtilis chromosome.We concluded that Dna K is the most efficient chaperone for rh FGF21 soluble expression in B.subtilis.5.Hecht et al.reported that rh FGF21 mutants(L98R,P171 A and P171G)retain their natural biological activity but display an improved resistance to aggregation and proteolytic cleavage in vivo.The result indicated that the inclusion body fraction was marginally decreased in L98 R of rh FGF21.6.During the post-log phase in B.subtilis growth,the bacteria express and secrete large amounts of protease,making secreted heterologous proteins difficult to accumulate due to protease hydrolysis.As the host strain B.subtilis 1A751 is already deficient in two major extracellular proteases(npr E and apr E),we knocked out the remaining six genes that express extracellular proteases in B.subtilis 1A751: bpr,epr,Htr A,mpr,npr B,and vpr,to protect the target protein rh FGF21 from protease degradation.These results illustrate that the target protein rh FGF21 is degraded by extracellular proteases and that a protease-deficient strain can help maintain the stability of mature secreted rh FGF21.7.Eleven signal peptides(SPpho D,SPpel,SPywb N,SPlip A,SPprot A,SPywm C,SPdac B,SPnpr E,SPydd T,SPyoqm,SPyvce)from B.subtilis were screened,and SPdac B was chosen as the most suitable signal peptide for the secretion of rh FGF21 in B.subtilis.8.To further improve the secretion level,we screened several transporters by integrating them chromosomally into B.subtilis.In conclusion,the Sec Y and Sec YEG were the relatively efficient transport proteins for rh FGF21 production in B.subtilis.9.To further optimize rh FGF21 expression conditions,the concentration of maltose and induction temperature were studied and reported.The Kno6 cf strain containing inducible promoters was cultivated in SR medium.The results indicate that the experimental conditions for suitable induction were determined to be 0.2%(w/v)maltose and 37 °C.As a result of the present study,we have constructed a maltose-inducible expression system for the expression and secretion of the heterologous protein rh FGF21 in B.subtilis.Through optimization and combination of effective factors,the secretion of rh FGF21 can reach 14% of extracellular total protein,and the soluble expression of rh FGF21 can reach 22% of total cell protein by shake flask fermentation.