Chemistry 3131 – Biochemistry Laboratory
Fall 2014
Purification of the Sco protein from Thermus thermophilus
Background
The Sco protein from Thermus thermophilus (TtSco) is a protein that is involved in the assembly
of cytochrome c oxidase, complex IV of the
respiratory system. The CuA site is a binuclear
copper center (Figure 1) that is the electron acceptor
from cytochrome c (1).
Figure 1: CuA site of cytochrome c oxidase
The Sco protein is thought to aid in assembly of the
CuA site of cytochrome c oxidase. Its exact function
is still the subject of much study, but at least one
function is to serve as a thiol-disulfide
oxidoreductase (2). A thiol-disulfide oxidoreductase
is a protein that will transform thiols and disulfide
bonds in a target protein. When the CuA protein is
synthesized, there is no copper in the binding site,
Figure 2: Proposed role of TtSco in the formation of the Cu A metal binding site.
and the adjacent cysteines are thought to form a disulfide bond. Sco is thought to reduce the
disulfide bond (Figure 2). Then a separate protein, called PCuAC, delivers the copper ions to the
metal binding site (2).
TtSco has been cloned in the Hunsicker-Wang laboratory and
put into a vector in which two purification tags, a histidine tag
and an S•tag, were added to the N-terminus of the protein. The
purification tag that we will utilize is the 6 sequential histidines,
the his-tag. This purification tag allows the protein to be
purified using Immobilized Metal Affinity Chromatography
(IMAC). In this case, the column is a Ni-NTA column, in which
the Ni2+ is bound to the resin through a nitrilotriacetic acid
Figure 3: Ni-NTA binding
(NTA) chelating group. The chelating group occupies 4
site in the resin (4)
coordination sites, leaving two weakly bound waters on the Ni2+
(Figure 3). The histidine tag interacts with the Ni2+, replacing the water ligands, and binding the
protein to the resin (Figure 4). The principle with this type of chromatography is that the tag
provides a way to selectively bind the protein to the resin while the rest of the will flow through.
Then the protein is eluted with a histidine mimic, imidazole (3-4) (Figure 5).
Figure 4: Histidine tag binding to the Ni-NTA resin (figure
taken from reference 4)
Figure 5: The structure of Histidine versus
imidazole
One consideration in this chromatography is that there will be some ion exchange characteristic
to the resin, since the resin terminates in a positively charged group. Thus, the buffer conditions
often include a higher ionic strength condition in order to screen the effect of the resin charge (34). In addition, a lower concentration of imidazole is maintained even in the equilibration buffer
to compete with any weakly binding proteins.
Ni2+ is not the only metal that can be used for IMAC. Successful reports include use of Zn2+,
Co2+, Cu2+ and Fe3+. The affinity of the protein for these metals can be used to optimize the
interaction of the protein and the resin (3).
References
(1) Ferguson-Miller, S., Babcock, G. T. (1996) Chem. Rev. 96, 2889-2907
(2) Abriata, L.A., Banci, L., Bertini, I., Ciofi-Baffoni, S., Gkazonis, P., Spyroulias, G.A., Vila,
A.J., Wang, S. (2008) Nat. Chem. Biol., 4, 599-601
(3) Scopes, R.K. (1994) Protein Purification: Principles and Practice, 3rd Ed., Springer, New
York, NY
(4) The Qiaexpressionist: Handbook for high-level expression and purification of 6xHis-tagged
proteins (2003) Qiagen
Sequence Alignment Instructions
There are many different alignment programs and databases to search to find proteins that are
related to your protein of interest. You will be using a set of web-based resources to find the
sequence and several related sequences that you use in the visualization. You will develop a
sequence alignment and use a program to make a .pdf of your sequence alignment. The
following are general guidelines on how to make a figure from a sequence alignment.
1. Go to www.pdb.org and search for the PDB ID or keyword search box. On the right side of
the structure summary, there is an option to download files. Choose FASTA sequence. (This is
just a format of text that other programs can read.) That should open a .txt file with the
information in it. Copy the sequence information to the clipboard.
2. Go to the NCBI website, http://www.ncbi.nlm.nih.gov/ This site is a collection of tools and
databases for biological molecules. Go to the BLAST link in the dark blue banner just under the
title. Hit BLAST. Go about half way down the site to protein BLAST. Paste your FASTA
sequence from the pdb in the “Enter query sequence” box. Click the BLAST button at the bottom
of the page.
3. Under Description, as you go down the page, there are other proteins that come up with
similar sequences or are found with enough homology for them to be listed. Their overlap with
the queried sequence is reflected in the E value. A smaller number is a better match. As you go
down the page, you will see another set of descriptors that show the alignment between the
queried protein and the protein found through the BLAST search.
4. At the very bottom of the page, click “Download”. This will take you to a new page. Choose
the FASTA option under the Display menu. This will put your chosen sequences that were
found in the BLAST search into the FASTA format. To save this file, under the Show menu,
chose, to text.
5. Copy the text. You will want to delete extra information of each FASTA sequence after the <
in order for your alignment to have descriptive line titles. Go to
http://www.ebi.ac.uk/Tools/msa/clustalo/. This is a program that will align all of the sequences
that you copied. Paste the FASTA format sequences from 5. In the window at the bottom of the
page that says “Enter or paste a set of sequences in any supported format”. (This is why the
sequences needed to be in FASTA format)
6. Click the run button. Once the alignment is done, a new page comes up. There is a lot of
information in this page. Even in this form, you can see the alignment of the sequences. *
indicates strictly conserved within the sequences.
7. Click the “Download Alignment” link (you may need to select “open in a new window or
tab”. This will save a .clustal file which can be read by Espript.
8. Now, we will use another web based program to make a ‘pretty’ figure. This program is
called Espript and is one of many possible programs. Go to
http://espript.ibcp.fr/ESPript/ESPript/index.php
9. To start the figure making, click the “Run ESPript 3.0” button at the top of the page. Load
your .clustal file in the “Main alignment file” box. This will give the program the alignment file
that you just made. Then hit submit.
10. There should be a window that appears that has the pdf file of the sequence alignment. Save
the .pdf file.
Protein purification report
Your post-lab should include results from the purification of the protein, the analysis using SDSPAGE gel electrophoresis and the concentration of the purified protein. In addition, you will
include the following items:
Molecular visualization:
Using your skills with chimera, include a ribbon diagram of the Sco protein from Thermus
thermophilus as a figure. The figure should show a ribbon diagram of Sco (PDB ID: 2K6V).
There are 31 structures in this file, so use only file 1 (#0.1). Show the 2o structure as cartoon
colored by structure (no side chains visible) using the default colors. Show Cys 47 and 51 from
the model #0.1 as ball and stick. These are the cysteines thought to be involved in the thiol
reductase activity. Include this picture in the results section of the report.
Sequence Alignment:
You will perform a sequence alignment of the Thermus thermophilus Sco protein. Be sure to
include comparison to human Sco1 (taxid 9606) (PDB ID: 2GGT), Yeast Saccharomyces
cervisiae (taxid 4932) (PDB ID: 2B7J), and Bacillus Subtilis (taxid 1423) (PDB ID: 1ON4) in
the sequences with a total of 5 sequences used to compare to TtSco. You can obtain the
sequences from the pdb or through the BLAST search (use only chain A if there are multiple
chains). If you use the BLAST search, you will need to specify the organism (under “choose
search set” beneath the query box) in order to obtain the 3 listed above. For the remaining two
sequences, make sure that you are choosing two other sequences that are not the Thermus
thermophilus Sco, since that is the protein that you are querying.
The sequence for TtSco is:
His tag
MHHHHHHSSG
PVDFALEGPQ
IFVSVDPERD
DHTATTFVVK
LVPRGSGMKE
GPVRLSQFQD
PPEVADRYAK
EGRLVLLYSP
TAAAKFERQH
KVVLLFFGFT
AFHPSFLGLS
DKAEATDRVV
Start TtSco
MDSPDLGTDD DDKMLPRGHT FYGTRLLNPK
RCPDVCPTTL LALKRAYEKL PPKAQERVQV
GSPEAVREAA QTFGVFYQKS QYRGPGEYLV
ADLQALL
Compare only the sequence starting with MLPRGHT… The amino acids before are the artificial
purification tags. Note that when you make the alignment figure, you will want to have the
designations for each protein to have a recognizable name. To do this, when you paste the
sequences in for the ClustalW alignment, make the first word after the > sign the name that you
want, i.e. human or Thermus. When you are finished making the figure using Espript, you will
want to save the file as a tiff or jpeg file that you can insert into your word document.
Discussion Questions (for purification, SDS-PAGE, visualization and sequence alignments)
1. How do the OD 595 nm measurements correlate with the presence of your purified protein?
2. Comment of the purity and yield of your purified protein. In the H-W lab, yields of this
protein are 5 mg pure protein / 1 mL lysis. Does your yield track with this value? If not,
postulate why.
3. What kind of homology does TtSco have with other proteins (high or low)? Given that TtSco
has been shown to be a thiol-disulfide oxidoreductase, would you expect the other Sco proteins
to have the same function? Could there be other functions?
4. Relate the 3-dimensional positions of the conserved cysteine residues to their thiol-disulfide
oxidoreductase activity.
5. PAGE gels can also be run as native gels. Why might one need to run both a native and an
SDS-PAGE gel? What alterations to the system must be incorporated?
6. How will the purity of the protein sample affect the concentration determination?
Fall 2014
Purification of the Sco protein from Thermus thermophilus
There is a Ni-NTA column pre-poured for your use. There are three buffers that will be used:
Equilibration Buffer (100 mM Imidazole, 50 mM Na2HPO4 pH 7.0, 300 mM NaCl, 0.05%
tween)
Elution Buffer (250 mM Imidazole, 50 mM Na2HPO4 pH 7.0, 300 mM NaCl, 0.05% tween)
Imidazole Wash Buffer (500 mM Imidazole, 50 mM Na2HPO4 pH 7.0, 300 mM NaCl, 0.05%
tween)
Notes: Be sure to record all the volumes of your fractions for your post-lab analysis.
1) Equilibrate the column with 10 mL of Equilibration buffer.
2) While the column is equilibrating, set aside 20 µL of the lysis solution for use in the SDSPAGE gel and Bradford analysis. Label it accordingly and store in the -20o C freezer.
3) Once the column is equilibrated, let the buffer level come to the same height as the top of the
resin. Close the stop cock. Align the column over the first tube.
4) Without disturbing the top of the resin, load 200 µL of the lysis solution. Open the stop cock
and allow the solution to penetrate the resin. Once the solution level is at the top of the resin,
turn off the stop cock.
5) Add 10 mL of equilibration buffer to the column, being careful not to disturb the resin. Open
the stop cock and collect 5 mL fractions in falcon tubes. Allow the buffer to go through the
column and be collected in the tubes. Continue until the buffer level reaches the top of the resin.
Turn off the stop cock. Mark the last tube that you used as ‘end wash’
7) Label the next tube ‘start elution’. Add 5 mL of elution buffer. Open the stop cock and
collect 1mL fractions in eppendorf tubes. Allow all of the buffer to flow through the column
until the buffer level reaches the top of the resin. Turn off the stop cock.
8) Mark the last tube used as ‘end elution’. Add 5 mL of the Imidazole wash buffer and open
stop cock. Collect 2.5 mL fractions until the buffer level reaches the top of the resin. Turn off
stop cock. Mark the first tube used as ‘start imidazole wash’ and the last tube used as ‘end
imidazole wash’
9) Wash the column with 10 mL of highly polished water.
10) Wash column with 20% ethanol by using 20 mL (you may need to add this in two steps).
Discard the ethanol flow through.
11) Label the samples with your group’s name. Store the samples in the refrigerator until next
week.