Last year, the iGEM Team Hamburg (Finding Chlamydori) worked on and modifying the receptor tar-envZ. This receptor is registered in the iGEM data bank as a fusion protein of the tar-domain and the intracellular EnvZ-domain, although the receptor has an aspartate-binding domain consisting of 484 amino acids. 255 amino acids belong to the N-terminal Tar-domain and 229 amino acids to the C-terminal EnvZ-domain. The assembled construct is 1516 base pairs long and registered as BBa_C0082. from the iGEM Team Antiquity.

The function of both domains has already been thoroughly investigated Utsumi et al. [1] und Mise et al. [2], thereby only the periplasmic aspartate-binding domain (amino acids 26 to 193) has been expressed and studied. In the original paper by Mise et al. [2], the Tar-domain was amplified out of the genome of Escherichia coli DH5a with primer pair 5‘ – ATG GCT AGC GAT GAC GAC GAC AAG GGC AGC CTG TTT TTT TCT TC – 3‘ (forward) and 5‘ – CTC GAA TTC TCA TTA TTG CCA CTG GGC AAA TC – 3‘ (reverse), and cloned in the expression vector pET-28a(+). In addition to the N-terminal start methionine, the vector has a thrombine cleavage site upstream of the C-terminal his-tag. According to the paper, tar domain was purified using the His-tag. His-tag renders the protein aggregation-prone, thus it has been removed afterwards through the thrombine cleavage site. The non-tagged protein was used for a crystallography.

The team last year did not investigate this part further. Instead, the Tar-EnvZ-receptor was cloned in an OmpR/GFP reporter construct and the GFP concentration measured after a mDAP induction of cells that were transformed with the whole construct. The results were published (shown here), although the yields might have been higher, if the aggregation propensity has been known before.

Our goal this year was to investigate whether the his-tagged tar-protein aggregate completely or only to a certain degree and how this can be prevented. We used the expression vector pET-28a(+) with N-terminal methionine and C-terminal His-tag for both the Tar-domain from Mise et al. and the biobrick BBa_C0082. Instead of only using the sequence 26 to 193 amino acids, we used the entire 255 amino acid sequence of the tar-receptor of the Tar-EnvZ-receptor. We used the following primers:

Forward primer: 5'-ATT GCA GCT AGC ATG ATT AAC CGT ATC CGC GTA GTC ACG CTG TTG G-3'

Reverse primer: 5'-ATT GCA GGA TCC TGA AAC GCT CTG CGC CAG GTC GC-3'

The product is 765 base pairs long (255 amino acids) and has a molecular weight of 31.2 kDa. With this primer pair and a DNA sample of the biobrick BBa_C0082 provided by the iGEM Headquarters we amplified the domain using the flowing PCR reaction: