Question 1

An insect expression system uses a baculovirus that is engineered to encode the protein of interest. The virus is used to infect ovarian cells from Spodoptera frugiperda, the fall armyworm. The advantages of using this system to produce engineered proteins are _________.

Accepted Answer

The insect expression system using baculovirus offers several advantages, including correct post-translational modifications, the ability for cells to grow in suspension in a simple broth, cell lysis at the end of the viral life cycle to release the protein, and high expression levels of the engineered protein.

Question 2

The advantages of using a yeast expression system are due to _______.

Accepted Answer

Yeast expression systems offer several advantages, including a fast growth rate, the ability to secrete proteins into the growth media, the capability of producing large quantities of engineered protein, and lower costs compared to mammalian expression systems.

Question 3

Proteins expressed in bacterial cells ________.
I. fold properly if they contain several domains
II. have to be released from the cells during a cell lysis step
III. do not go through post-translational modifications
IV. are secreted into the growth media

Accepted Answer

Proteins expressed in bacterial cells often do not undergo post-translational modifications like those in eukaryotic cells, because bacteria lack the cellular machinery for most of these modifications. Additionally, to isolate and purify the proteins expressed in bacterial cells, the cells typically need to be lysed to release the proteins, as they are not usually secreted into the growth media.

Question 4

Engineered proteins can be expressed in a variety of host cells (mammalian, bacterial, etc). Choosing the host cell depends on ___________.

Accepted Answer

The choice of host cells for expressing engineered proteins depends on factors such as the growth rate of the cells, the need for specific post-translational modifications for the protein's biological activity, the complexity of the protein (single-domain or multi-domain), and the quantity of protein required. Each of these factors can influence the efficiency, cost, and feasibility of protein production, making them all critical considerations in the selection process.

Question 5

Using site-directed mutagenesis, the mutation of a serine residue to an alanine residue might be used to _________.
I. decrease the solubility of a protein
II. eliminate the possibility of phosphorylation at that location
III. observe the impact of a non-polar side chain at that location
IV. prove that serine had a catalytic function in the native protein
V. promote proper folding of the protein

Accepted Answer

Serine to alanine mutation eliminates the possibility of phosphorylation at that location (II) because alanine cannot be phosphorylated. This mutation also allows observation of the impact of a non-polar side chain at that location (III) since alanine is less polar than serine. Additionally, if the mutation affects the activity of the protein, it could prove that serine had a catalytic function in the native protein (IV).

Question 6

It is common, when making mutations in a cDNA sequence ___________________.

Accepted Answer

When making mutations in a cDNA sequence, it is common to use a codon with the smallest change in the nucleotides to minimize the impact on the protein function while achieving the desired mutation.

Question 7

Adding restriction sites when constructing plasmids will help with _______.

Accepted Answer

Adding restriction sites to plasmids is crucial for cloning because it allows for the insertion of foreign DNA into the plasmid at specific locations, facilitating the construction of recombinant DNA molecules.

Question 8

When using cassette mutagenesis, in order to insert an engineered cDNA sequence into a plasmid, a __________ is used to cut out the sequence that is no longer needed, and a __________ is used to insert the new mutant cassette into the plasmid.

Accepted Answer

Restriction enzymes are used to cut DNA at specific sequences, allowing for the removal of unwanted sequences. Ligase is then used to insert and join the new sequence (mutant cassette) into the plasmid.

Question 9

PCR-based mutagenesis is a widely used technique due to the mutation being introduced in _________.

Accepted Answer

The answer of PCR-based mutagenesis is a widely used technique...

Question 10

Mutation of a serine residue to a(n) _____ residue is a good modeling tool for a phosphorylated serine.

Accepted Answer

Aspartate is a good modeling tool for a phosphorylated serine because it has a similar negative charge and can mimic the electrostatic interactions of the phosphate group.

Question 11

__________ suppression is used to incorporate unnatural amino acids into a protein using cellular expression.

Accepted Answer

The answer of __________ suppression is used to incorporate unnatural...

Question 12

Subtilisin is a bacterial serine protease that is commonly used as an additive in laundry detergent. In its native state, it has a methionine that becomes oxidized, surprisingly leading to enzyme inactivation. To prevent inactivation, subtilisin could be engineered by mutating methionine residues to ___________.
I. isoleucine
II. alanine
III. cysteine
IV. phenylalanine

Accepted Answer

Methionine can be oxidized, leading to enzyme inactivation. Replacing it with non-oxidizable residues like isoleucine, alanine, or phenylalanine can prevent this.

Question 13

The green fluorescent protein (GFP) ___________.
I. is a large protein, MW larger than 60 kDa
II. is used in chimeric protein constructs
III. is extremely useful in tagging proteins inside the cell, to visualize the localization of the expressed proteins
IV. was first isolated from the jellyfish Aequorea victoria
V. needs a substrate to exhibit fluorescence

Accepted Answer

II, III, and IV are correct. GFP is not a large protein (it's around 27 kDa), and it does not need a substrate to exhibit fluorescence; its chromophore forms spontaneously.

Question 14

Proteins can be engineered to express a tag that is beneficial in __________ of the protein by _______ chromatography.

Accepted Answer

The answer of Proteins can be engineered to express a...

Question 15

Choose fusion proteins from the list that could be used in biochemical or cellular assays:
I. GFP-protein constructs
II. extra cellular matrix proteins
III. anti-His6 tag-antibody conjugated to horseradish peroxidase
IV. maltose binding proteins
V. subtilisin

Accepted Answer

Fusion proteins like GFP-protein constructs, anti-His6 tag-antibody conjugated to horseradish peroxidase, and maltose binding proteins are commonly used in biochemical or cellular assays for purposes such as visualization, purification, and detection.

Question 16

His6-tagged proteins are often overexpressed in E. coli cells and purified by _______ chromatography.

Accepted Answer

Nickel-affinity chromatography is commonly used for the purification of His6-tagged proteins because the histidine residues have a strong affinity for nickel ions, which can be immobilized on the chromatography resin.

Question 17

In the cell, most proteins fold without assistance and within milliseconds. The information needed to fold a protein properly is found in the ______ sequence of the protein.

Accepted Answer

The primary sequence of a protein, which is its sequence of amino acids, determines how the protein will fold into its functional three-dimensional structure. This sequence dictates the interactions between amino acids that lead to the protein's final shape.

Question 18

Protein folding is the process by which proteins ____________.
I. are stored
II. gain their secondary structure
III. gain their tertiary structure
IV. lose activity
V. burry hydrophobic side chains and expose hydrophilic ones

Accepted Answer

Protein folding is the process through which proteins gain their secondary and tertiary structures, crucial for their functional conformation. During this process, hydrophobic side chains are typically buried inside the protein structure, while hydrophilic ones are exposed to the aqueous environment, aiding in the protein's proper function and solubility. This does not inherently involve storage or loss of activity, but rather is a critical step in achieving a functional protein structure.

Question 19

Levinthal's conclusion was that protein folding _______, because it would take way too long to complete the folding if all randomly available rotations around the main chain bonds would be sampled to achieve the most stable structure.

Accepted Answer

The answer of Levinthal's conclusion was that protein folding _______,...

Question 20

During folding the entropy of the protein _______.

Accepted Answer

During folding, the entropy of the protein decreases because the protein is transitioning from a less ordered state (unfolded) to a more ordered state (folded), which means there is a reduction in the number of possible configurations or microstates the protein can adopt.

Quiz 20: Designer Proteins and Protein Folding

An insect expression system uses a baculovirus that is engineered to encode the protein of interest. The virus is used to infect ovarian cells from Spodoptera frugiperda, the fall armyworm. The advantages of using this system to produce engineered proteins are _________.

The advantages of using a yeast expression system are due to _______.

Proteins expressed in bacterial cells ________. I. fold properly if they contain several domains II. have to be released from the cells during a cell lysis step III. do not go through post-translational modifications IV. are secreted into the growth media

Engineered proteins can be expressed in a variety of host cells (mammalian, bacterial, etc) . Choosing the host cell depends on ___________.

It is common, when making mutations in a cDNA sequence ___________________.

Adding restriction sites when constructing plasmids will help with _______.

When using cassette mutagenesis, in order to insert an engineered cDNA sequence into a plasmid, a is used to cut out the sequence that is no longer needed, and a is used to insert the new mutant cassette into the plasmid.