Abstract: Calvin-benson-bassham cycle-related enzyme (CbbX) is regarded as red-like RuBisCo activase that activates RuBisCo in Chromophyta.In Saccharina japonica, cbbX gene is encoded in nuclear genome (nuc cbbX) and plastid genome (pt cbbX).With RT-PCR, the fragment containing intact ORF of nuc cbbX gene and restriction enzyme cutting site was amplified in this study.Multiple sequence alignment carried out for the protein sequences of nucCbbX, ptCbbX of S.japonica and CbbX of Rhodobacter sphaeroides (Rs CbbX) revealed the predicted mature nucCbbX contained AAA+ domain, Walker-A and -B motifs and residues related to CbbX functions (ATPase, binding RuBP and keeping hexamer structure stability).The pairwise sequence similarities between nucCbbX and ptCbbX, nucCbbX and Rs CbbX were 37.4% and 36.8%.While the similarity between ptCbbX and Rs CbbX was relatively high 57.6%.The predicted 3D structure of nucCbbX contained three subdomains which were N-terminal extension consisting of one α-helix, α/β subdomain containing 5 α-helixs and 5 β-foldes, and C-terminal 5-helix bundle subdomain.For further study the crystal structure of nucCbbX, the target fragment was then successfully subcloned into the express vector pET28a and was expressed in E.coli BL21(DE3) induced with 1% IPTG.The result of SDS-PAGE showed that the fusion protein expressed mainly in the form of inclusion bodies with molecular weight of 50.2 kDa which is greater than that of nucCbbX mature protein 44.7 kDa.The fusion proteins were purified after being denatured by urea.And the purified proteins were proven that they could be used in protein crystallization after renaturation.

Key words: Saccharina japonica, Nuclear genome, CbbX, Protein, Expression and purification

CLC Number: 

• Q78