Proteins

• polymers of Amino Acids
• Specific shapes
• specific functions
• structure- function relationships

• Enzymes- biological catalysts - speeds up chemical reactions
• Hormones - chemical messengers (sends signals)
• Antibodies - proteins that bind and neutralize foreign molecules (immune system)

• Involved in many production processes
• -Chymosin: an enzyme involved in producing casein (milk/cheese protein) -- food processing
• textlies/leather goods; Creation of the wool/leather, stone-washed jeans
• detergents; proteases lipases amylases

Erythropoietins; stimulate blood production (people with anemia; kidney disease)

Insulin; used to treat Type 1 diabetes ( for peple who can't regulate glucose pancreas)

• Used to treat disease
• a number are now on the market
• Humira TM; Adelimumab, used to treat rheumatoid arthritis and other conditions
• -TNF inhibitor ( an anti-inflammatory agent)
• -A "blockbuster" drug ( >$1 billion in sales/year)
• antibodies attack foreign tissue; protein

• structure determines function
• four levels of protein structure
• If protein doesn't fold properly it wont work properly

• a long polymer of amino acids
• a protein is a functional polypeptide that might have other things associated with it

• The linear amino acid sequence of the polypeptide
• 20 common amino acids
• a change in 1 amino acid in the sequence might make the protein lose all function

• the primary structure folds , just barely, into alpha helices or beta pleated sheets
• 

• the polypeptide folds into a specfic 3D structure
• certain attractions are present between the alpha helices and pleated sheets causing the bending and twisting into shape
• 

• More than 1 polypeptide chain associates in 3D structure
• lustering
• of several individual peptide or protein chains into a final
• specific shape. A variety of bonding interactions including hydrogen
• bonding, salt bridges, and disulfide bonds hold the various chains
• into a particular geometry. There are two major categories of proteins with quaternary structure - fibrous and globular.
• 

• Two phases
• Upstream processing (getting proteins to express) {proteins are produced by cells genetically engineered to contain the human gene
• which will express the protein of interest}
• Downstream processing (recovery and purification of biosynthetic products)

• Microorganisms
• -Bacteria
• -fungi (yeast)
• Advantages
• -cheap and easy to grow in large fermentors
• Problems
• - Bacteria don't modify mammalian proteins "correctly" (don't fold properly)
• bacteria don't put sugar on cells, if sugar is important use mammalian cells
• Neither do yeast, but they are a little better

• Insect cells - sugars are still not the same as mammalian cells.
• Mammalian cells ex; CHO cells , Human embryonic kidney (HEK)
• Advantages - proper processing of proteins
• Disadvantages - Expensive to grow -Difficult to scale up (bioreactors)

• Farming
• Transgenic goats or cows - insulin in cows; milk cows for insulin using a milk promoter
• Transgenic plant -tobacco

• For secreted protein, media is collected or
• Whole cells are broken open

• Keep protein extract cold
• remove or inhibit proteases ( chop iup protein)
• Don't create foam, minimize shear forces
• (gentle handling ) dont shake

• protein precipitation (add salt for concentration to increase, protein falls out of solution to the bottom of tube, becomes white , you must know the concentration your protein alone will respond to. After removing soln add media to precipitation to dissolve.
• Selective precipitation can remove proteins that arent the one you want, or preciptate yours (and then redissolve and renature)

• Size-based separation (holea so small it can separate proteins from each other)
• centrifugation
• Membrane filtration
• microfiltration
• Ultrafiltration

• Several methods commonly used
• Often done in a column; then called column chromatography

• SEC
• Gel filtration chromatography
• *Tiny beads with tiny holes
• separates proteins by size
• Smallest proteins travel fastest through the column
• hplc= high pressure chromotography

• IEC
• Separates proteins by charge
• Different types of IEC resins separate proteins differently (but using same principles)
• * column with a positive charge the negative ones will stick to it and the positive charge will go through

• Very specific- relies on ability of a protein to specifically bind a substance (or antibody)
• Terminology
• Ligand : a molecule that specifically binds something els ( protein)
• Ex: use an antibody against your protein
• Pass a mixture over the AB column -Only your protein sticks; everything else passes through
• Wash column
• elute your protein

• Another technique that can separate a mixture of proteins
• Native gel electrophoresis -separates proteins based on charge
• SDS-PAGE (SDS poluacrylamide gel electrophoresis) -separates proteins based on size
• Isoelectric focusing(IEF) - separates proteins based on isoelectric point

Combine methods in a process that separates your protein from the rest of them without inactivating your protein

Used to check purity of proein (among other uses)

• High performance liquid chromatography
• Chromatography (already covered) UNDER PRESSURE (faster)
• FPLC (fast performance LC) * not as good as HPLC but faster

• Mass Spec or MS(measures the mass of the protein)
• Very sensitive method to determine the mass of a small protein
• Lots of "alphabet soup" here
• - MALDI-TOF
• LC/MS, EC-MS, MS/MS

• combines 2 electrophoresis techniques
• IEF and SDS-PAGE
• (Sodium D SULFATE) strongly negative molecule
• that sticks across protein and straightens it out to be able to measure its size
• Proteins with more positive charge will migrate to bottom and Vice Versa

• Lyophilization -freeze-drying
• Store in solution - will likely have to be kept at 4 deg C
• Freezing - Not the best way to package your protein
• A rule of thumb is to limit freeze-thaw cycles to 3