| Chondroitin sulfate?CS?,a kind of acidic linear chain polysaccharide,is composed of glucuronic acid?GlcA?and N-acetyl-galactosamin?GalNAc?.And,CS is the best drug for preventing and treating joint diseases and repairing the damaged central nervous system,and widely used in food and cosmetics field.While,the applications of CS is varied with its structure.For example,Chondroitin sulfate A?CSA?and Chondroitin sulfate C?CSC?are mainly used in the treatment of osteoarthritis.Therefore,the preparation of single structure of CS is particularly important for the development of clinical applications and new functions.Previous studies have shown that in vitro enzymatic catalysis produce single structure of CS.Among them,Chondroitin-4-O-sulfotransferase-1?C4ST-1,EC 2.8.2.5?catalyzes the sulfation of 4-OH of GalNAc to generate CSA.In E.coli BL21?DE3?and P.pastoris GS115,the expression level of C4ST-1 was low and could not be applied to clinical production.So,the expression level was optimized in this study.In E.coli BL21?DE3?,the effects of three protein tags and two molecular chaperones?Erv1p and DsbC?were investigated for the soluble expression of C4ST-1.And the soluble protein tags,signal peptides,and constitutive promoters on the expression of C4ST-1 were also investigated in P.pastoris GS115.The main conclusions of this study are as follows:?1?The soluble expression of C4ST-1 in E.coli BL21?DE3?was improved based on the N-terminal fusion strategy and co-expression of Erv1p and DsbC.The C4ST-1 mutants,fused with three protein tags?MBP,SUMO,TrxA?,were expressed in E.coli BL21?DE3?by DNA recombination techniques.The results showed that the recombinant strain with N-terminal fusion of TrxA had the highest enzyme activity,which was 9.78±0.45 U·L-1.To further increase the expression level of intracellular soluble protein,we investigated the effects of co-expression of thiol oxidase Erv1p and disulfide isomerase DsbC.The results showed that co-expression of DsbC significantly increased the expression level of intracellular soluble protein(21.99±0.42U·L-1,2.33-fold).While no significant effect was observed when co-expression Erv1p(12.32±0.76 U·L-1,1.30-fold).Then,C4ST-1,Erv1p and DsbC were co-expressed and the enzyme activity in shake flask and 3 L fermenter was improved to 29.12±0.66 U·L-1 and49.97±0.73 U·L-1,respectively.?2?Optimized promoters increased constitutive expression of C4ST-1 in P.pastoris GS115.Based on the Gibson assembly strategy,four constitutive promoter(PGAP,PPGK1,PTEF1,PGCW14)were ligated with the linearized vector pPIC9K-?-SUMO-C4ST-1 to substitute PAOX1,respectively.Under the control of four different constitutive promoters,the recombinant P.pastoris strains were expressed,in which PTEF1 was the best promoter for constitutive expression.After 96 h fermentation,the enzyme activity was reached to 50.70±0.51 U·L-1.Therefore,PTEF1 can be used as a potential promoter for constitutive expression of C4ST-1 in P.pastoris GS115.?3?The secretion expression level of C4ST-1 in P.pastoris GS115 was improved based on the N-terminal optimization strategy and optimization of culture conditions.In order to increase secretory expression level,two protein tags?SUMO,TrxA?and seven signal peptides??,???57-70?,??VAVE?,SUC2,P18,nsB,PHO1?were compared.The results showed that when the N-terminal of C4ST-1 fused with SUMO tag and added VAVE amino acid sequence behind the KEX2 cleavage site of?-factor signal peptide,the extracellular enzyme activity of C4ST-1 increased from 7.07±0.30 U·L-1 to 43.05±0.31 U·L-1;By optimizing the conditions of the flask cultivation,the enzyme activity of C4ST-1 reached to 62±0.58 U·L-1,which was1.42 times than parent.Further,the enzyme activity of C4ST-1 reached 189.00±2.08 U·L-1through high density fermentation in 3 L fermentor. |
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