In vitro expression of phytoplasma immunodominant membrane proteins

Strategies for in vitro expression in Escherichia coli of Imp immunodominant membrane proteins from 'Candidatus Phytoplasma asteris' chrysanthemum yellows strain (CY) and "flavescence doree" (FD) phytoplasmas are reported. The entire imp genes of CY and FD were cloned, sequenced and aligned with homologous sequences of phytoplasmas of different phylogenetic groups. Partial CY and FD imp gene sequences devoid of the transmembrane domains were cloned into the pQE32 and pRSetC vectors, but following IPTG induction, both fusion Imp proteins were expressed at low levels. To increase protein expression of the partial FD Imp, codon usage of the gene and accessibility of translation initiation were modified according to that of E. coli. The new construct showed 80% nucleotide similarity with the original FD imp gene, identical amino acid sequence and improved expression level. A second protein (about 28 kDa) was eluted under the same conditions as the putative partial His-tagged FD Imp, but the latter protein was the only specifically recognized by an antihistidine antibody in Western blot. These results are the first indication that, at least for FD, even in the absence of the troublesome transmembrane domain, an optimized codon usage is useful to achieve in vitro expression of immunodominant protein.