Polyhistidine variety environments making it a highly versatile

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
proteins.

GFP are used as reporter gene,
reporter gene is used to allow researchers to see if the gene of interest is
expressed in an organism. GFP is a good reporter gene because it glows green fluorescence
under UV lights when it is attached to the DNA of the gene of interest,
reporting very clearly if gene is active in the organism. GFP is composed of
238 amino acid residues, in the center of GFP a single chromophore made of a
single chain of four amino acids that is responsible for fluorescence colour
without needing any cofactors. Because chromophore is well protected inside the
cylindroid shape, therefore GFP can fluoresce in variety environments making it
a highly versatile protein.

We Will Write a Custom Essay Specifically
For You For Only $13.90/page!


order now

The appropriate site of positioning
of GFP during protein fusion is at the primary sequence of the protein of
interest, domains are tried to be avoided. GFP can be attached to both N
terminal or C terminal of protein of interest, but typically one end of the
protein contains the function domain that could be sterically hindered by
addition of GFP, therefore GFP will be attach to the opposite end. If a protein
contains signal sequence at the N terminal, and a retention sequence at C
terminal, GFP will be placed immediately after the signal sequence and before
the retention sequence, therefore it will be less likely to interfere with the function
of either sequence. If the protein of interest is transmembrane protein, GFP
cannot be attached near the transmembrane domain.

GFP displays two absorptions maxima
at neutral form 397nm and anionic form 477 nm, blue to ultraviolet range, the
emission is peak at 509 nm, light green range. Many different mutants of GFP
have been engineered due to the evolving needs of researchers; in particular,
blue fluorescent protein (BFP),
cyan fluorescent protein (CFP), and yellow fluorescent protein derivatives (YFP).

 

Nostoc punctiforme is a species of
filamentous cyanobacteria, only type of prokaryotes able to produce oxygen. Researchers
have used green fluorescence protein reporter to construct two transcriptional
reporter shuttle vectors for Nostoc punctiforme. In Escherichia coli host,
ampicillin and kanamycin resistant plasmid abled promoters to clone into the
multiple cloning sites. The N. punctiforme was tested by cloning promoters
expressed in vegetative cells, nitrogen fixing heterocysts and akinetes. GFP
driven from promoter hetR was found highly expressed in heterocysts, whereas GFP
driven from promoter psaC reporter gene fusion shown expression in
vegetative cells but not in heterocysts, GFP for N.punctiforme homologue
akinete specific gene avaK was expressed in the development of akinetes. These
results demonstrates the utility of GFP vecotrs for studying cell type specific
gene expression in differentiating filamentous cyanobacteria.