Chapter 5Proteins: Primary Structure

Biochemistry IDr. Loren Williams

Revised 2/14/2014

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).

Protein Purification

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 inhibitorsTreat tubes and surfaces with

albuminControl the pH with buffers

illustration of cell lysis

from biofreak(http://bio-ggs.blogspot.com/)

Protein Purification

Figure 5-5

Protein Purification

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.

Protein Purification

Protein PurificationMethods 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]

Protein Purification

Bovine erythrocytes under osmotic stress

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Protein Purification

Protein Purification

Separate your protein from other cellular components.

Ammonium sulfate precipitationProteins 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.

Protein PurificationChromatography

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.

Protein Purification

Protein Purification 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 C-18, 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.

Protein PurificationSize Exclusion Chromatography

Figure 5-7

Size Exclusion ChromatographyProtein Purification

Protein Purification

Figure 5-6

Ion Exchange ChromatographyProtein Purification

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 S-transferase), His (hexahistidine)….

- To study cellular localization or detect your protein easily, use fluorescent tag like GFP.

Green Fluorescent Protein

Protein Purification

Maltose Binding Protein:

The vector pMAL (a plasmid) is designed to create maltose-binding 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.

Protein Purification

Pmal System (MPB tag): SDS-polyacrylamide gel electrophoresis of fractions from the purification of MBP-paramyosin-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.

Protein Detection absorption

Figure 5-4

Protein Detection fluorescence

Figure 5-9

Protein Detection (SDS PAGE)

Protein Detection staining

Figure 5-3

The enzyme-linked immunosorbent assay (ELISA) is a test that uses antibodies and color change to identify a substance.

Table 5-2

Omit from here on, spring 2014

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

Omit 2013

Figure 5-16a

Omit 2013

Figure 5-16a part 1

Omit 2013

Figure 5-16a part 2

Figure 5-16b

Omit 2013

Figure 5-17

Omit 2013

Figure 5-18

Figure 5-19

Table 5-4

Figure 5-20

Figure 5-23

Figure 5-24