Transcript 5 Protein Primary Structure - School of Chemistry and Biochemistry

Revised 2/14/2014
Biochemistry I
Dr. Loren Williams
Chapter 5
Proteins: Primary Structure
A protein is one or more large polymers consisting of linear
chains of amino acids.
Proteins perform a vast array of functions within living organisms,
including:
(i) catalyzing chemical reactions,
(ii) regulating reactions,
(iii) transport and sequestration
(iv) pores and channels
(v) structure (stiffness and rigidity),
(vi) tensile strength,
(vii) compartments and adhesives
(viii) signaling molecules and hormones
(ix) optical devices
Proteins differ from one another primarily in their sequence of
amino acids, which usually results in the folding of the protein
into a specific three-dimensional structure that determines its
activity.
Gelatin is a colorless, tasteless, water-soluble derivative
of collagen of cow or pig. Made from bones, hides and
connective tissues. Collagen doesn't dissolve in
waterThe body parts are ground and treated with strong
acid or strong base, then boiled, then dried and ground
to a powder.
Gelatin is contained in jello, skittles, gummy bears,
starbursts, marshmellows...
There are 2,598,960 combinations of 5 cards in a 52 card
deck.
There are 400 billion stars in the Milky Way.
There are a around a septillion stars (1024) in the
universe.
There are 10130 possible protein sequences of 100 amino
acids in length (20100)
Table 5-1
Bovine Insulin
Insulin is a hormone made of polypeptide (i.e., it is a protein) that
is produced by the pancreas. Insulin central to regulating
carbohydrate and fat metabolism in mammals.
Table 5-5 part 1
Table 5-5 part 2
Figure 5-21
Figure 5-22
 Decide on a protein source: Whale myoglobin is easy to
get (in Japan). Human transcription factors are low copy
number and can only be obtained by over-production by
recombinant methods (usually in E. coli).
 Develop methods to detect the protein (absorbance at
280 nm, catalytic activity, gel mobility, fluorescence, etc).
Myoglobin was the first protein whose three-dimensional structure was
determined by x-ray diffraction. The experiments used a lot of purified
protein, which at that time could not be produced by recombinant
methods. Myoglobin is the primary oxygen-carrying protein of muscle
tissues. Myoglobin is particularly abundant in marine mammels, allowing
the animals to hold their breaths for long periods.
Break open (lyse) the cells.
Keep the cell lysate on
ice
Add protease inhibitors
Treat tubes and
surfaces with
albumin
Control the pH with
buffers
illustration of cell lysis
from biofreak
(http://bio-ggs.blogspot.com/)
Figure 5-5
You hope that your
over-expressed
protein remains
soluble in the E. coli.
But sometimes it will
form inclusion
bodies.
Inclusion bodies are
dense particles of
aggregated foreign
protein caused by
high-level
expression.
Methods for cell disruption
 Osmotic Pressure
 Detergents
 Beadbeating
[tiny hard beads and intense agitation by stirring or
shaking]
 Sonication [Ultrasound (20–50 kHz)]
 French Press
[A piston forces a pressurized sample through a needle
valve, causing shear stress and decompression]
Bovine erythrocytes under osmotic stress
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Separate your protein from other cellular components.
Ammonium sulfate precipitation
Proteins can be separated from a mixture on the
basis of relative hydrophilicity by gradually increasing the
concentration of ammonium sulphate. Add ammonium
sulfate and pellet cellular proteins by spinning in a
centrifuge. Discard the supernatant, resuspend the protein
and redisolve with detergents. Then remove the ammonium
sulfate and detergent by dialysis.
Chromatography
Flow a solution containing the
protein through a column
containing resin. Proteins can
interact selectively with various
types of resins, and can thus be
separated by the time required to
pass the column, or by the
conditions required to elute the
protein from the column.
 Size exclusion chromatography: The resin contains pores.
Smaller proteins enter pores and migrate more slowly.
 Hydrophobic interaction chromatography (reverse phase): The
resin contains a hydrophonic material (silica modified with C18, etc). Elute by decreasing the aqueous content of the solvent
and increasing the ethanol.
 Ion exchange: The resin is charged. Anionic resins are
positively charge retain negatively charged proteins. Cationic
resins hare negatively charged and retain positively charged
protein. Protein can be eluted by high salt.
Size Exclusion Chromatography
Size Exclusion Chromatography
Figure 5-7
Ion Exchange Chromatography
Figure 5-6
Affinity Tagging and Protein Over-Expression
Fuse the tag to your protein: There are many different tags available.
- To express and purify a protein use MBP (maltose binding protein), GST (gluthatione Stransferase), His (hexahistidine)….
- To study cellular localization or detect your protein easily, use fluorescent tag like GFP.
Green Fluorescent Protein
Maltose Binding Protein:
The vector pMAL (a plasmid) is designed to create maltosebinding protein (MBP) fused to any target protein.
A gene or open reading frame is inserted into a restriction site of
the vector polylinker, in the same translational reading frame as
the malE gene (encoding maltose-binding protein).
The fusion protein produced in E. coli is generally stable and can
be purified by amylose affinity chromatography (cheap and easy).
After purification the fusion protein the protease Factor Xa
separating the target protein from the MBP.
Pmal System (MPB tag):
SDS-polyacrylamide gel electrophoresis
of fractions from the purification of MBPparamyosin-ΔSal. Lane 1: Protein Ladder
(NEB #P7703 ). Lane 2: uninduced cells.
Lane 3: induced cells. Lane 4: purified
protein eluted from amylose column with
maltose. Lane 5: purified protein after
Factor Xa cleavage. Lane 6: paramyosin
fragment in flow-through from second
amylose column.
absorption
fluorescence
Figure 5-4
Figure 5-9
staining
The enzyme-linked
immunosorbent assay (ELISA)
is a test that uses antibodies
and color change to identify a
substance.
Figure 5-3
Table 5-2
Figure 5-8
Figure 5-11
Figure 5-12
Box 5-1
Figure 5-13
Figure 5-13 part 1
Figure 5-13 part 2
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Page 107
Page 107
Table 5-3
Figure 5-14
Figure 5-14 part 1
Figure 5-14 part 2
Figure 5-14 part 3
Figure 5-15
Figure 5-15 part 1
Figure 5-15 part 2
Figure 5-16a
Figure 5-16a part 1
Figure 5-16a part 2
Figure 5-16b
Figure 5-17
Figure 5-18
Figure 5-19
Table 5-4
Figure 5-20
Figure 5-23
Figure 5-24