protein 1

Question 1

What are some different types of column chromatography for protein separation?

Answer 1

1. Gel filtration/size exclusion chromatography
2. Ion exchange chromatography
3. Hydrophobic interaction /reverse phase chromatography
   4.Affinity chromatography

Question 2

What are some techniques of protein detection by immunoassays?

Answer 2

1. Western blot
2. Immunoprecipitation (IP)
3. Enzyme-linked immunosorbent assay (ELISA)
4. Lateral flow Immunodiffusion

Question 3

Name some functions of proteins in a cell

Answer 3

 Enzymes
 Structural
 Transport
 Motor
 Storage
 Signaling
 Receptors
 Gene regulation

Question 4

What is the origin of the word ‘protein’?

Answer 4

Greek word proteios, meaning the “first place”.

Question 5

How many amino acids are found essentially in proteins of all living organisms on earth?

Answer 5

20 ALPHA amino acids

Question 6

Name some categories that amino acids can be divided into?

Answer 6

1. Nonpolar (hydrophobic)
2. Polar (hydrophilic)
3. acidic
4. basic

Question 7

What are the 4 levels of organization of proteins?

Answer 7

Primary
Secondary
Teritiary
Quaternary

Question 8

What is meant by primary structure of protein?

Answer 8

sequence of amino acid chain; one dimensional

Question 9

What is meant by secondary structure of protein?

Answer 9

amino acid strand acquires a spring-like shape as they repel and attract eacher other; 2 dimensional

–> alpha-helix, beta pleated sheets

Question 10

What is meant by tertiary structure of protein?

Answer 10

the coiled strand of AA folds and loops over on itself to take on a functional shape
–> three dimensional

Question 11

What is meant by quaternary protien structure?

Answer 11

a protein that consists of one or more AA strands.

Once coiled and folded the protein can function as it is or may join other proteins, or add carbohydrates, vitamins or minerals

Question 12

Proteins have a variety of shapes, surface charges and sizes.

True or false

Answer 12

True

Question 13

Proteome are far more complex than genome. Why?

Answer 13

alternative splicing, mRNA editing and post-translational modifications give rise to the complexity of proteome

Question 14

How are protein structures determined mainly?

Answer 14

by crystallographic analysis of pure
crystalized proteins

Question 15

Name a database where structural info of proteins is deposited.

Answer 15

PDB protein databank.

Question 16

Name a high resolution structural protein database of
coronavirus.

Answer 16

COV3D

Question 17

What is the advantage of having a databas with info regarding coronavirus?

Answer 17

identify spike structural classifications
identify sequence diversity

Question 18

When were insulin products first introduced to market?

Answer 18

in 1923 by Lilly

Question 19

How is insulin produced today?

Answer 19

genetically engineered bacteria produce animal-free insulin

Question 20

How was insulin obtained in the 1950s?

Answer 20

10,000 pounds of pig pancreases made 1 pound of insulin

Question 21

What is the insulin produced by Eli Lilly called?

Answer 21

humulin

Question 22

Give some examples of biologics

Answer 22

• monoclonal antibodies
• recombinant hormones/proteins
• cell therapy

Question 23

What are biologics?

Answer 23

a class of drugs that are produced using biological processes rather than being chemically synthesized.

–> derived from living organisms such as humans, animals, microorganisms, or plants

Question 24

What are the top 5 pharma companies in the world?

Answer 24

1. pfizer
2. johnson and johnson
3. abbvie
4. merck
5. roche

Question 25

Name some anti-TNF-a therapeutics

Answer 25

- remicade (infliximab) - humria (adalimumab) - Enbrel (etanercept)

Question 26

What are the 3 methods of recombinant protein expression?

Answer 26

transformation transduction transfection

Question 27

State the differences between transformation, transduction and transfection?

Answer 27

Transformation: prokaryotes; non-viral Transduction: prokaryotes or eukaryotes viral methods/vectors Transfection: eukaryotes; non-viral methods

Question 28

What are some commonly used expression hosts for target gene cloning?

Answer 28

1. bacteria expression systems (E. coli) 2. yeast expression systems 3. insect expression system (silkworm) 4. mammalian expression systems

Question 29

What are popular mammalian cell lines for recombinant protein expression?

Answer 29

1. Human embryonic kidney 293 cells 2. Chinese hamster ovary (CHO) cells

Question 30

How were HEK 293 cells derived?

Answer 30

from a spontaneously aborted fetus or HEK cells grown in tissue culture taken from a female fetus

Question 31

What is the downside of using HEK 293 to produce recombinant proteins?

Answer 31

more costly

Question 32

What is HEK 293T?

Answer 32

derivative human cell line that expresses a mutant version of SV40 large T antigen

Question 33

What is the advantage of using CHO cells to produce recombinant proteins?

Answer 33

1. low human virus cross contamination 2. easily developed and high yield of secretory complex proteins at lower cost

Question 34

What is chromatography?

Answer 34

a wide ranges of laboratory techniques to separate a mixture of components based on differences in partition of individual component between the two phases, the stationary(solid) phase and mobile phase

Question 35

Briefly explain the steps in chromatography

Answer 35

1. mixture to be separated is dissolved in mobile phase 2. mobile phase is added throughout the process 3. components separate 4. each component is collected as it reaches the bottom of the column

Question 36

What kind of chromatography are proteins best separated by?

Answer 36

column chromatography packed with different solid phase materials by taking advantage of different modes of partitions.

Question 37

What is paper/thin layer chromatography used for?

Answer 37

- for quick qualitative analysis of small organic molecules such as phytochemicals *Little use in the large scale purification of complex biomolecules such as proteins DNA

Question 38

What property is hydrophobic interaction chromatography (HIC) and reverse phase chromatography (RPC) based on?

Answer 38

hydrophobicity

Question 39

What property is Ion exchange chromatography aand chonotofocusing based on?

Answer 39

charge

Question 40

What property is gel filtration/size exclusion chromatography based on?

Answer 40

size

Question 41

What property is affinity chromatography based on?

Answer 41

biorecognition (ligand specificity)

Question 42

What is the principle of size exclusion chromatography?

Answer 42

1. sample injected 2. size separation 3. larger molecules elute prior to smaller molecules 4. chromatogram

Question 43

What is the principle of ion exchange chromatography?

Answer 43

1. column equilibrium 2, sample adsorption 3. sample desorption

Question 44

Name some anion exchangers in IEC.

Answer 44

quaternary ammonium (strong) diethylaminoethyl, DEAE (weak)

Question 45

Name some catanion exchangers in IEC.

Answer 45

Sulfopropyl (strong) Methyl sulfonate (strong) Carboxymethyl (weak)

Question 46

What are the steps in IEX chromatography?

Answer 46

1. IEX medium is equilibrated with start buffer (low ionic strength buffer) 2. Oppositely-charged proteins bind to ionic groups of IEX medium. uncharged or ones with same charge elute 3. ionic stregth increase to displace bound proteins --> gradient elution 4. wash 5. re-equilibration

Question 47

What is the separation of proteins in hydrophobic interaction chromatography (HIC) and reverse phase chromatography (RPC) based on?

Answer 47

hydrophobic properties of proteins

Question 48

How are proteins eluted in HIC? What starting buffer is used?

Answer 48

-starting buffer in high salt concentration i.e. 2M ammonium sulfate -elution by reducing salt concentration by weakening hydrophobic interaction with the solid support

Question 49

In Hydrophobic interaction chromatography (HIC), how does reducing the salt concentration weaken the hydrophobic interaction?

Answer 49

high salt concentrations can shield these hydrophobic regions from water molecules, effectively enhancing the strength of hydrophobic interactions between the target molecules and the stationary phase.

Question 50

When is RPC typically used?

Answer 50

for high resolution separation of peptides prior to mass spectrometric analysis

Question 51

In what order are proteins separated in HIC and RPC?

Answer 51

in order of increasing surface hydrophobicity

Question 52

What kind of separation can affinity chromatography be used for?

Answer 52

fusion protein purification

Question 53

What is a fusion protein?

Answer 53

proteins that are artificially created by combining the coding sequences of two or more genes for instance: protein of interest + tag/partner protein

Question 54

What type of chromaography is used for fusion protein separation?

Answer 54

affinity chromatography

Question 55

What is the principle of affinity chromatography?

Answer 55

based on specific high affinity interaction of immobilized ligand/receptor with the target proteins

Question 56

What is protein A chromatography?

Answer 56

used method for the purification of antibodies --> high affinity of Protein A for the Fc region of immunoglobulins --> highly specific and efficient antibody purification

Question 57

For protein A chromatography, protein A media can be based on:

Answer 57

1. polysaccharides 2. silica 3. polymers 4. magnetic particles

Question 58

Name some commonly used affinity purification tags.

Answer 58

1. His6 2. GST (glutathione S-transferase) 3. MBP (maltose-binding protein) 4. FLAG 5. BAP 6. Strep-II 7. CBP

Question 59

What is IMAC (immobilized metal affinity chromatography)?

Answer 59

for the purification of proteins based on their affinity for certain metal ions (His6 is used as tag) --> metal ions immobilized on solid support, selectively bind proteins containing specific amino acid residues, such as histidine tags (His-tags)

Question 60

What metal ions could be used for IMAC?

Answer 60

nickel (Ni²⁺), cobalt (Co²⁺), or zinc (Zn²⁺) typically attached to chelating groups like nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA).

Question 61

How are bound proteins eluted in IMAC?

Answer 61

by competition with free histidine or by using imidazole or other chelating agents that disrupt the metal-protein interaction

Question 62

What is the advantage of IMAC?

Answer 62

1. high selecivity 2. Affinity-based Purification 3. Mild Elution Conditions: 4. High Binding Capacity

Question 63

Why use solubility enhancing fusion partners?

Answer 63

for facilitated purification

Question 64

What happens if the fusion protein is not soluble?

Answer 64

1. Expression Issues 2. Purification Challenges 3. Loss of Functionality 4. Poor Protein Stability

Question 65

Give some examples of slubility enhancing fusion partners.

Answer 65

MBP maltose-binding protein GST glutathione-S-transferase

Question 66

Name some commonly used proteases for tag removal

Answer 66

Enterokinase Factor Xa Thrombin TEV HRV TagZyme

Question 67

Name the tag for IMAC

Answer 67

His6

Question 68

What is the advantage of using both His 6 and GST as tag?

Answer 68

His6 --> initial purification using IMAC GST --> soluble and for serve as cleavage site for both tags

Question 69

In the fusion protein construction strategy, what type of fusion proteins could be expressed?

Answer 69

1. His tag for IMAC 2. epitope tag for immunoaffinity column 3. ligand-binding domain for conventional affinity column 4. precipitation domain for non-chromatographic method 5. compound tag for simultaneous detection and purification

Question 70

What is strep-tactin?

Answer 70

high-affinity streptavidin derivative used for protein purification and detection --> based on the specific interaction between Strep-Tactin and the Strep-tag.

Question 71

How to elute biotin-labled protein from streptavidin?

Answer 71

use desthiobiotin as a competitor