Download Hes1 Gene: Cloning, Protein Expression and Structural Study

Hes1 Gene: Cloning, Protein Expression and Structural Study
Kumar J. R.1 and Swaminathan K.2
Department of Biological Sciences, National University of Singapore
Blk S2, 14 Science Dr 4, Singapore 117543.
ABSTRACT
Lymphoma is the fifth most common cancer in the USA. (American Cancer Society) Hes1 is
a significant signal effector in the signalling pathway allowing the proliferation of lymphocytes.
It regulates the transcription of other genes through various novel mechanisms some known,
others being researched upon. Being a consequential molecule, its own activities are regulated
by other proteins being investigated. This project targeted to analyse the Hes1 protein structure
through X-ray crystallography and perhaps progress towards a possible drug for cancer. The
research methodology involved isolating the gene through the amplification; forming a
recombinant plasmid vector; transforming bacterial cells with the recombinant vector;
expressing the protein and optimising the conditions for expression. Several other steps for the
purpose of verification of the gene sequence as well as protein were also carried out. Difficulties
in amplifying and expressing the entire gene lead to the eventual amplification the truncated
DNA-binding domain of the Hes1 gene. Despite a frame-shift mutation in the amplified gene
sequence, the protein expressed seemed to be of an appropriate size. Further confirmation could
be carried out in the form of His-tag binding or mass spectrometry.
INTRODUCTION
Hes 1 is a regulator that is involved in the control of several pathways which are important in
the proliferation of the cell. Thus the mechanisms of control adopted by Hes1 are well-studied.
Being an important multi-functional transcriptional controller, the regulation of its own
transcription has been well-researched. However, the protein structure of Hes 1 has the protein
structure of Hes1 has not been profoundly analysed. This research project aimed to clone the
Hes1 gene into a modified vector and express the protein; eventually examining the protein
structure by X-ray crystallography. In the future, an antibody blocking Hes 1 protein can be
designed allowing it to serve as a cancer drug.
MATERIALS AND METHODS
3 types of primers were constructed for isolating the gene coding whole protein (WP), the
DNA binding domain(region of protein that binds to DNA to control transcription) and Orange
domain (structural feature that has been conserved among its family of transcriptional
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regulators). In all 3 types of primers, the forward contained the EcoR1 restriction site and the
reverse contained the Xho1 site. PCR was performed 5 times to achieve good clones made
visible through gel electrophoresis. 2 different cell lines (SUPM-2 & SUDHL-2) were used,
altering annealing temperatures. These clones were then extracted from the gel using the
protocol of a PCR purification kit. The purity and concentration of the clones was checked using
a spectrophotometer.
These GOI clones and pET-M vector were then double digested using EcoR1 and Xho1
enzymes. The double digested vector was run on gel and extracted from the gel; its purity and
concentration checked. The 2 double digested GOI and vector are then ligated. These ligated
samples were transformed into DHP-α cells by inducing competency through a heat shock.
These cells were plated on ampicillin (Amp) medium and allowed to grow. Colonies were
picked at random to undergo colony PCR. T7 promoter and terminator primers were used.
Comparative analysis between the negative control and samples allowed the colonies with
recombinant plasmids to be identified.
Sequencing PCR was then carried out. The colony with the recombinant plasmid was first
inoculated in a culture medium. After which the curing of the plasmids was conducted by
following the protocol of a Plasmid Quick kit. These plasmid samples then underwent PCR
using either the T7 promoter or terminator as primers. They were then sent for sequencing.
Small-scale protein expression was then conducted by first transferring the transformed
plasmids to BL-21 cells. These cells are inoculated in culture tubes containing Luria-Bertani
broth and Amp. After the optical density has reached 0.6, these cells are induced with different
concentrations of Isopropyl β-D-1-thiogalactopyranoside (IPTG). The cells are then sonicated
and centrifuged separating soluble proteins and insoluble proteins. These protein samples are
subjected to SDS-PAGE. A faint band at 10kD is observed. Western blotting is used to transfer
the proteins to a membrane and an antibody that binds to the His-tag present in the POI is used
to probe and identify the presence of the POI.
RESULTS AND DISCUSSION
Eventually clones of the DNA-binding domain and Orange domain were obtained from the
SUPM-2 cell line at annealing temperatures between 55-65oC. Their purity and concentration
were checked to ensure they were good enough to be used for the further experiments.
Double digestion is done to ensure that the GOI was not inserted into the vector in an
inverted manner. This would prevent protein expression. It also helped to ensure that the vector
did not re-anneal after being digested and different sticky ends would be present. DHP-α cells
are bacterial cells specialised for taking up the recombinant plasmids due to more porous cell
walls, absence of plasmids and it also has the benefit of being non-pathogenic thus, facilitating
its use in the laboratory. The polylinker sites in the vector are situated within the T7 site which
can be transcriptionally activated by gratuitous inducer IPTG. Hence in a recombinant plasmid
the GOI is located within the T7 site. Thus the T7 primers used in colony PCR should amplify
the GOI in the recombinant plasmid as opposed to just the T7 site in the negative control. This
leads to a difference in size.
The sequencing results showed a frame-shift mutation which resulted in a premature
termination codon preventing the last 19 amino acids from being translated. However, the
sequencing results arrived after protein expression had been performed.
BL-21 cells are specialised for protein expression as they are protease-deficient. IPTG acts as
an inducer removing repression of the Lac operon in which the GOI is present allowing the
Protein of Interest (POI) to be over-expressed. Despite the mutation a 10kD protein is detected
in the soluble protein samples through SDS-PAGE which is not present in the negative control.
The mutation should have caused the protein to be smaller whereas the actual size of the protein
is around 10.3kD. Western blotting allows the proteins to be transferred to a membrane which
binds proteins. Milk protein binds indiscriminately to the rest of the membrane (excluding the
areas where the POI has already binded) and prevents the antibody from binding to the
membrane. Hence the primary-antibody from the mouse, binds to the tag of 6 histidines in the
protein. Being anti-mouse, the secondary-antibody binds to the primary-antibody. The reporter
enzyme attached to secondary-antibody that catalyses a reaction product that is
chemiluminescent and hence darkens sensitive X-ray film enabling concentration-dependant
detection. The Western blot was unsuccessful. The positive control did not show a band;
perhaps the antibody was defective. Further tests (his-tag purification, mass spectrometry) can
be conducted to confirm the identity of the protein. After the protein has been identified X-ray
crystallography can elucidate protein structure and eventually be used to design an antibody.
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