1How are AAs generally removed from the body?
Amino groups are turned into ammonia and excreted as Urea
2What are the two steps (in order) to convert an amino acid into a keto acid and ammonium?
Transamination
Deamination
3What enzyme catalyzes the transamination step?
Aminotransferase
4What enzyme catalyzes the deamination step?
Glutamate Dehydrogenase
5What are keto acids used for?
ATP, Glucose, and AA synthesis
6Why is ammonia toxic?
Ammonia can cross the blood-brain barrier, leading to the formation of glutamic acid (a-ketoglutarate + NH3). The citric acid cycle is inhibited and ATP production declines.
7What is the overall goal of the urea cycle?
Converts highly toxic ammonia into urea for excretion.
8How much ATP is consumed in one urea cycle?
3 ATP
9What are glucogenic amino acids?
Amino acids whose carbon skeletons are degraded into pyruvate, a-ketoglutarate, succinyl-CoA, fumarate, or oxaloacetate, which can be used for glucose synthesis.
10What are ketogenic amino acids?
AAs whose Carbon skeletons degraded into acetyl-CoA, which can be converted into ketone bodies or fatty acids.
11Which type of AAs, Ketogenic or Glucogenic can NOT be used for synthesizing glucose?
Ketogenic
12What 5 AAs are both glucogenic and ketogenic?
Trp, Tyr, Phe, Thre, ile
13What are the only two ketogenic amino acids?
Lys and Leu
14What is an essential amino acid?
An amino acid that cannot be synthesized in the body and must be supplied in the diet.
15What is a non-essential amino acid?
An amino acid that can be synthesized in the human body.
16Where can Glu and Gln be synthesized in the citric acid cycle?
From a-Ketoglutarate
17What is Phenylketonuria (PKU)?
Abnormal metabolism of Phe because of a deficiency of Phe hydroxylase, causing phenylpyruvate buildup in the body.
18Accumulation of phenylpyruvate in infants leads to?
Severe intellectual disability in infants.
19Those suffering from PKU should not consume high levels of Phe. This artificial sweetner should not be consumed because it contains Phe.
Aspartame
20What are the three general steps to recombinant protein expression and purification?
1. Transfect cells with a DNA vector (plasmid) that contains the template.
2. Culture cells to transcribe and translate the desired protein.
3. Isolate and purify the desired protein.
21What is a plasmid?
A small and circular DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently.
22What is the origin of replication?
A sequence at which replication is initiated
23What is a selectable marker?
A marker that enables selection of the transformed cells.
24What imparts resistance to antibiotics?
The selectable marker
25What is the T7 Promoter of a plasmid?
A region that binds to the T7 RNA polymerase to initiate transcription of the gene of interest.
26What is the Lacl gene?
A gene that codes for the lac repressor.
27What is the SPRTN coding sequence?
A gene that codes for the SPRTN protein.
28What is the origin (ori)?
The starting point for replication
29Explain the simple mechanism for plasmid transformation?
Bacteria and Plasmid (encoding whatever) are mixed.
Ca2+ is added to bring bacteria and plasmid close together.
Heat Shock to get plasmid into the cell.
Grow cells on plate of choice.
30How does IPTG trigger recombinant protein induction?
IPTG binds to a lac repressor, causing the dissociation of the lac repressor and operator, then the T7 RNA polymerase binds to the promotor and the transcription is initiated.
31What device is used to cause cell lysis by disrupting the cell membrane?
Sonicator
32What is the role of the HHHHHH tag?
This is a tag at the N or C terminus that binds to Ni2+ to separate out for purification purposes.
33Ion-exchange affinity is based on what?
The isoelectric point
34How does ion-exchange chromatography work?
Separates protein based on their change. A higher salt concentration causes everything to elute as the ions in the column are blocked.
35What is an isoelectric point?
The pH of a solution at which the net charge of a protein is zero.
36Negatively charged proteins bind to what exchanger?
Anion Exchanger
37Positively charged proteins bind to what exchanger?
Cation Exchanger
38If the purification buffer has a pH of 7.5 what ion-exchange chromatograpgy should be used to bind SPRTN (pI = 8.99)?
pH < pI Protein = Positively Charged
39How does size exclusion chromatography work?
Different protein sizes means that proteins will pass through a column and elute at a different time.
40T or F: The smaller a protein, the longer a path, and longer time until elution.
True
41What is protein engineering?
The modification of a protein with recombinant DNA technology to get a better use in medicine, industry, agriculture, and others.
42What are the three basic steps in protein engineering?
1. Modify the DNA sequence through a mutation
2. New protein is expressed with mutation modification
3. Does the protein have improved stability and functional protein? If yes yay. If no trash.
43What is the most common method for causing gene mutations?
Radiation exposure (UV) causes a cross-link between pyrimidine bases. The errors are produced during DNA replication by repair enzymes.
44What is the limitation for radiation-caused mutagenesis?
Inefficient and out of control.
45How does PCR work?
1. Denature a template strand.
2. Anneal with added DNA primers, nucleotides, and DNA polymerase.
3. Synthesize new strands
4. Repeat cycle
46What is site-directed mutagenesis?
Plasmids from E. coli cells are methylated. The PCR-amplified DNA is not methylated.
47What is DpnI in site-directed mutagenesis?
An enzyme that eats only methylated DNA
48What is the limitation of site-directed mutagenesis?
Limited Scope
49What is error-prone PCR?
Changing the PCR conditions to make the PCR process prone to errors during amplification.
50What 4 methods can be used to change the PCR conditions, leading to more errors during amplification?
Low Fidelity Polymerase
Staggered dNTP concentrations
Add MnCl2
More PCR Cycles
51What is the error rate for Taq polymerase? What about Pfu polymerase?
1/1000;
1/10000000
52What is DNA shuffling?
The digestion of a gene by DNaseI into random fragments, and the reassembly of those fragments into a full-length gene by primerless PCR.
53In DNA shuffling, are primers necessary?
No
54How does DNA shuffling work?
DNaseI cuts random spots on DNA forming DNA fragments. Annealing and extension of fragments occur and amplification occurs.
55How does one determine the purity of a recombinant protein?
Polyacrylamide gel electrophoresis (PAGE)
56How does one confirm protein identity from a mixture?
Western Blot
57How does one determine the concentration of a purified protein?
Bradford Assay
58How does one clean a protein sample?
Dialysis
59What is native PAGE commonly used for?
Separating proteins based on size and charge.
60How do proteins move through native PAGE?
Larger proteins get tangled in the mesh more, and travel slower.
61For PAGE analysis, what is used to reduce disulfide bonds?
2-mercaptoethanol
62Why use SDS in PAGE analysis?
SDS destroys the tertiary structure and coats the proteins in negative charges.
63How are proteins separated in SDS-PAGE?
Size ONLY
64What is Bromophenol Blue?
An electrophoretic color marker used to indicate when the gel is done.
65What is the role of Coomassie Blue R-250?
This binds to R, K, H producing the blue color that is seen in the gel.
66Between native PAGE and SDS-PAGE, which method keeps proteins in a 3D structure?
Native
67How are proteins separated in SDS-PAGE vs Native PAGE?
SDS: Size ONLY
Native: Size and Charge
68What is western blot methods used for?
Detecting a specific protein in a mixture of proteins.
69What are the six steps involved in western blot techniques?
1. SDS-Page
2. Transfer Proteins to membrane
3. Block membrane with BSA
4. Add Primary Antibody
5. Add Secondary Antibody
6. Signal Production
70What is the purpose of blocking the membrane with BSA?
Block interfering noise from the membrane.
71What is the purpose of the primary antibody?
To bind to the protein
72What is the purpose of the secondary antibody?
Bind to the primary antibody and contain an enzyme conjugated to it.
73What is the purpose of the enzyme conjugated to the secondary antibody?
Consume the substrate and produce a signal.
74What is the purpose of the Bradford assay?
To measure the concentration of total protein in a sample.
75Binding of this dye results in a color change from brown to blue.
Coomassie Dye
76What does Coomassie dye bind to?
Basic AAs (Lys, Arg, His)
77How is the concentration determined in the Bradford assay?
Prepare a standard curve with BSA.
Measure the unknown sample absorbance
Use y = mx + b to calculate the sample concentrations.
78What is diffusion?
Movement of molecules from high to low concentration until equilibrium is reached.
79How does dialysis work?
Molecules small enough pass thorugh the pores of a semipermeable membrane diffusing into a buffer solution whereas the larger molecules remain in the dialysis bag.
80You have protein X = 2000MW and Protein Y = 1200 MW. when combined together they make a complex. What size dialysis membrane would be best for separating the proteins so that only the complex remains?
Use a semipermeable membrane with a size >2000MW but less than 3200MW, ideally closer to 3000.
81What is UniProt used for?
This is a comprehensive resource for protein sequence and annotation data
82What is EXPASY used for?
Calculating MW and pI of a protein.
83What is PDB?
Database of protein structures.
84What is PyMOL used for?
Visualizing Protein Structures
85What is Clustal Omega used for?
AA, Polypeptide, Protein Sequence Alignment