Lecture 4

Question 1

5 areas where protein is localised in Ecoli and features

Answer 1

cytoplasm - largest. Where protein synthesis occurs because it’s where ribosomes are.

cytoplasmic membrane - phospholipid bilayer. Cell already has mechanisms to transport proteins here because it does for it’s normal transport, membrane and resp proteins

periplasmic space - proteins with disulphide bonds expressed here.

Outer membrane - phospho lip, used to transport proteins

Secreted - completely outside cell in extracellular medium

Question 2

3 different protein types

Answer 2

soluble
cytoplamsic (inner) membrane proteins
outer membrane proteins

Question 3

properties of soluble proteins

Answer 3

hydrophillic surface
many diff folding structures - but always hydrophobic bit in middle
most found in cytoplasm (cos biggest capacity)
some found on periplasm

Question 4

properties of cytoplasmic membrane proteins

Answer 4

hydrophobic surface - cos in lipid environment
form alpha helices in transmembrane regions
hard to express at high levels

Question 5

why are cytoplasmic membrane proteins hard to express at high levels

Answer 5

highly regulated - Because hard to get them to the membrane - most attempts to get them there would result in cell damage so there is very strong regulation
(a reason why you induce protein expression in growth phase - high chance lots will die but hopefully enough around you get a good yield)

Question 6

properties of outer membrane proteins

Answer 6

hydrophobic

form beta barrels -often their function is to provide channels or present proteins on surface

Question 7

When do inclusion bodies occur

Answer 7

When proteins are expressed in the highest levels in the cytoplasm (biggest capacity), the protein comes together to form inclusion bodies - possibly because hydrophobic proteins can simply clump together without folding

Question 8

To make inclusion bodies a good way to produce lots of protein for easy purification, what must the proteins be able to do in vitro

Answer 8

be easily refolded

Question 9

How are proteins freed from inclusion bodies and refolded?

Answer 9

heat

add 6m urea slowly to bodies, proteins will naturally fold

Question 10

Who’s dogma is this method of freeing proteins from inclusion bodies based on and what does it state?

Answer 10

Alfinsen’s

That he protein has all the info it needs to fold correctly encoded in it’s own amino acid sequence.

Question 11

Give an example of a protein which is expressed in inclusion bodies and how it is successfully attained.

Answer 11

Proinsulin - lysozyme added, releases bodies, then given optimum refolding conditions in vitro.

Question 12

In what form do you need to express a protein if it won’t refold itself?

Answer 12

Active

Question 13

What is the role of chaperone proteins when expressed to improve the solubility of cytoplasmic proteins

Answer 13

help prevent aggregation

Question 14

Give examples of chaperone proteins

Answer 14

DnaK

GroEL

Question 15

Describe what chaperone proteins do to ensure proper folding

Answer 15

Recognises protein going down wrong route
forms a cavity inside which can accommodate misfolded protein
unfolds it
sets it on it’s way again
hopes that next time it will fold right

Question 16

What catalyses protein folding

Answer 16

ATP

Question 17

as well as chaperone proteins, what helps prevenmt aggregation when proteins are trying to fold?

Answer 17

The masking of the hydrophobic regions

Question 18

Can the ecoli cytoplasm form disulphide bonds? Why?

Answer 18

No - it’s a reducing environment

Question 19

Where are disulphide containing proteins found? Why?

Answer 19

The periplasm. Because it’s oxidising

Question 20

What’s the humidity?

Answer 20

?

Question 21

What enzyme catalyses insertion of disulphides?

Answer 21

DsbA

Question 22

What enzyme catalyses isomerisation of disulphides?

Answer 22

DsbC

Question 23

Describe the common process between both isomeristaion and insertion of disulphides

Answer 23

enzyme sits in oxidised form, acts as an intermediate and is reduced itself.

Question 24

is formation of disulphides oxidation?

Answer 24

Yar

Question 25

describe basically how the enzymes form the disulphide bonds

Answer 25

by removing the H’s from the S on the protein

Question 26

Give an example in the UK food industry where the presence of DsbA like protein during refolding enhanced activity of recombinant protein

Answer 26

when expressing chymosin

It converts casein to parcasein in curdling so used in cheese production

Question 27

How many disulphide bonds down chymosin have

Answer 27

3

Question 28

What are the 2 ways to make proteins with disulphide bonds

Answer 28

Express proteins in periplasm where disulphides form (call this option 1)
Make cytoplasm more oxidising (option 2)

Question 29

Describe how you’d go about doing option 1

Answer 29

add signal peptide to the protein - cleaved then just mature protein remains in peri

Question 30

2 problems with option 1

Answer 30

limited rate of export through the sec machinary - which is what transports proteins to peri
smaller capacity than cytoplasm

Question 31

2 examples of proteins made using option 1

Answer 31

human growth hormone

antibody fv fragments

Question 32

2 things you do to achieve option 2

Answer 32

remove thioredoxins and glutaredoxins - (enzymes which make cytoplasm oxidising)
Express DcsB in cytoplasm by removing it’s signal peptide so it doesn’t go where it normally would

Question 33

Example of proteins made by option 2 and how many disulphide bonds does it have

Answer 33

human tissue plasminogen activator

9. Whoa.

Question 34

Why fuse protein sequences to recombinant proteins

Answer 34

Stability

Question 35

Where can a fusion protein bind to on the recomb protein?

Answer 35

C or N terminus

Question 36

How is a fusion protein added to a recomb protein?

Answer 36

DNA added into expression vector

Question 37

Give an example of where a fusion protein has been added to the recomb protein somatostatin, including which end the fusion is added to and where it can be cleaved off

Answer 37

N terminal fusion of Beta-galactosidase.

Can be cleaved in vitro

Question 38

what is an alternative strategy to adding a fusion protein to increase stability?

Answer 38

Use a protease deficient ecoli strain to incr half life of protein

Question 39

Another use of fusions, as well as stability

Answer 39

Purification

Question 40

What is the most commonly used fusion tag for purification?

Answer 40

Hexa-histidine

Question 41

What type of matrix is on the column a protein with a hexa his residue is loaded on? (Once the lysate has been released by breaking open the bacterial cell)

Answer 41

nitrilotriacetic acid matrix (charged with nickel ions)

Question 42

name the 2 steps undertaken once the His tagged protein has bound to the column

Answer 42

wash - other proteins removed

elute - just his-tagged protein left

Question 43

Name 2 other fusion proteins and the columns on which they are purified

Answer 43

Glutathione-S-transferase - glutathione affinity column

Maltose binding protein (can target protein to periplasm as well - amylose resin column

Question 44

What does each fusion protein encode (generally), and what does this permit?

Answer 44

A sequence specific protease cleavage site located in region between affinity tag and cloned gene.
Permits removal of fusion protein tag following affintiy purification (normal purification, like previously mentioned)

Question 45

What 2 apparatus move recombinant proteins across inner membrane (in secretion)?

Answer 45

Sec or Tat apparatus

Question 46

What part of the secretory system spans both membranes of ecoli?

Answer 46

Type 1

Question 47

Are Ecoli good secretors?

Answer 47

No, they’re rubbish. But do have a mechanism to extrete toxins so exploit that

Question 48

How has ecoli been exploited to successfully incr inner membrane protein expression (normally inserting them there limits yield because lots of cells don’t survive it)?

Answer 48

Strains isolated with mutation for incr yields of membrane proteins (possible because this mutation has put an intracellular network within membranes so can transport recombinant proteins)