Date of Award
Master of Science (MS)
The Euryarcheota branch of the Archaea contains histone proteins that are highly homologous to eukaryotic histones. Virtually all species within this group have two histone proteins (designated H1A and H2B). These proteins contain a canonical histone fold motif that stabilizes homo- and perhaps hetero-dimeric interactions. Molecular modeling calculations previously carried out in our laboratory revealed that both homo and hetero-dimers of histones from two euryarchaeal organisms are equally plausible. We have also shown through molecular modeling that inter-species hetero-dimers are structurally identical to existing solution structures archaeal histones. An interesting question that needs to be addressed is whether homo- and hetero-dimeric species exist in the living cell and if they do, whether they are both physiologically functional. To address these questions we cloned the H2A and the H1A genes from Methanobrevibacter smithii (MS-H2A) and Pyrococcous furiosus (PF-H1A) in the polycistronic vector, pST44. When cloned individually in the pST44 vector, Escherichia coli BL21 (DE3) PLysS transformants were viable while E. coli BL21 (DE3) RIPL were not. However, when both genes were placed in pST44 and expressed simultaneously neither strain survived. Since expression of both genes is under the control of the LacI repressor increased protein expression should not be significant enough to account for cell death. It is more reasonable to suggest that hetero-dimer formation has led to the formation of a functionally distinct protein that is lethal in insignificant amounts (i.e. in the presence of LacR repression as well as PLysS repression). Furthermore, we successfully expressed both genes in the E. coli BL21 (DE3) PLysS. However, when attempting to purify the resulting histones after gene expression, the histones which contained affinity tags failed to bind to the corresponding affinity resin. We propose that this might be due to the possibility that the tags which are present on the N-terminus of the proteins are buried in a hydrophobic pocket that is formed by the proline-proline tetrad motif which exists in all archaeal histones.
Alqurashi, Doha Abdullah, "The cloning and expression of histone genes from the archaeal organisms Pyrococcus furiosus and Methanobrevibacter smithii in the polycistronic vector pST44." (2013). Electronic Theses & Dissertations. 134.