Spider dragline silk is a remarkably strong fiber with impressive mechanical properties,which were thought to be resulted from the specific structures of the underlying proteins and their molecular size.In this study,a silk protein 11R26 containing three repeats of the consensus amino acid sequence from the dragline silk protein of Nephila clavipes was used.To analyze the potential effects of the special amino acids on the function of 11R26,three protein derivatives ZF4,ZF5 and ZF6 were obtained by site-directed mutagenesis,based on the sequence of 11R26,and among these derivatives,serine was replaced with cysteine,isoleucine and arginine,respectively.After being expressed in Escherichia coli BL21(DE3)and purified by Ni column,all the four proteins reached the titer of 1.5 g/L in 5 L fermenter,with a similar molecular size of about 18.0 kDa.Subsequently,the mechanical performance of the fibers derived from the four proteins was tested.Both hardness and average elastic modulus of ZF4 fiber increased 2.2 times compared with those of 11R26.The number of disulfide bonds in ZF4 protein was 4.67 times to that of 11R26,which implied that disulfide bonds outside the poly-Ala region affect the mechanical properties of spider silk more efficiently.