Polyhistidine tags are histidine
residues chained together at both the N terminus or C terminus in the recombinant
protein coding sequence. Most common his-tag consist of six histidine residues
(hexahistidine), but a chain can be assembled of four to ten residues. There
are two ways to add his-tag. In a vector, the his-tag is added by inserting the
recombinant protein has the tag ready to fuse at the C terminus. Another way is
to perform polymerase chain reaction with primers that possess repetitive
histidine codons next to the Start or Stop codon. Placement of the tag on N or
C terminus depends on the protein of interest, the best tag position is after
determination of three-dimensional structure of protein and tag to not
interfere with protein’s domain, also his-tag must be exposed to exopeptidase during
purification. His-tag purification process is generally called affinity
purification or immobilized metal affinity chromatography (IMAC) technique. The
his-tag proteins have affinity and bind to the nickel ions attached to agarose
beads, other proteins do not have his-tag cannot bind with nickel ions, or
binds very weakly. Therefore, to separate his-tagged proteins from others, use wash
buffers and low concentrated imidazole to interfere with the weak binding
proteins. Then high concentration of imidazole is used to remove his-tagged

The life cycle of Cryptosporidium is complicated
consisting of both sexual and asexual developmental stages. Infection begins
with the ingestion by the host of the sporulated oocysts through contaminated
water or food or directly via the faecal-oral route. Inhalation of the oocysts
can also occur. Each oocyst contains four sporozoites which after excystation
in the intestinal lumen or the respiratory tract, emerge and invade the
epithelial cells and develop into trophozoites. Trophozoites undergo asexual
division (merogony) and form Type I Meronts consisting of 8 merozoites. Some of
these merozoites form Type II meronts which contain 4 merozoites and initiate
the sexual phase of the life cycle. Macrogametocytes and microgametocytes are
formed, fertilize and produce the zygote. Most of the zygotes develop into
oocysts, the thick ones with a two-layered wall which are released to the
environment, and the thin-walled oocysts 
which facilitate autoinfection (Thompson et al., 2005).


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