Roland Hilgarth

1
Roland Hilgarth (Sarge Lab) B106
BBSRB rshilg1_at_uky.edu 257-7349
2
Single Nucleotide Polymorphisms (SNPs)
3
Lots of Ways to Look at SNPs
4
Western Blotting
Method to use antibodies to detect expression of
mRNA as protein in the cell.
Involves separating protein on SDS-Page
electrophoresis and transfer of protein
electrophoretically to nitrocellulose or PDV
filters which are then probed with antibodies.
Sensitivity Can detect 0.1 ng of a 50 KDa
protein Approximately 1000 protein
molecules/cell Load capacity for gel approx.
150mg/well
5
Immunoprecipitation for Detection of
ProteinProtein
Interactions
Generally do a preclear with protein
G-agarose. Control reaction is just IgG
6
Yeast-2-Hybrid
Plasmids have different selectable markers
prey
bait
GAL4 promoter or LexA promoter
7
Common Features of Bacterial Expression Plasmids
Bacterial Origin of Replication Promotor/Operato
r RBS Multiple cloning site Transcription
Terminator Selectable Marker
8
Bacterial Protein Expression
Advantages
Drawbacks
Relatively Simple Cheap to Grow Flexibility Sop
histication
Lack Post-Translational Modifications Expressed
proteins can be in inclusion bodies (insoluble)
9
Some Common Bacterial Expression Protein Tags
Other Tags are under currently emerging such as
6-His SUMO and 6-His Ubiquitin Tags. There are
also tags such as FLAG that can be purified with
antibody columns
10
Bacterial Fusion Tags Generally Have
a Protease Cleavage Site to Allow for Tag Removal
Asp Asp Asp Asp Lys
Enterokinase Ile Glu/Asp Gly Arg
Factor Xa Protease Leu Val Pro Arg Gly Ser
Thrombin
11
To get Idea of Versatility Novagen pET vectors
12
Advantages and Disadvantages to Expression
Recombinant Proteins in Mammalian
Expression Systems
Advantages
Disadvantages
More expensive to do Technically more
challenging Take more time to do Difficult to
do on large scale
Proper protein folding Proper posttranslational m
odifications Recombinant proteins are almost
always accumulate in the correct cellular
compartment
13
Common Features of Mammalian Expression Vectors
Plasmid based
Bacterial origin of Replication Kozak
sequence Selectable Markers Constituitively or
Induce protein expression (promotor
driven) Produce large amounts of Protein
(Enhancer driven)
14
Common Features of Mammalian Expression Vectors
Bacterial origin of Replication Kozak
sequence Selectable Markers Constituitively or
Induce protein expression (promotor
driven) Produce large amounts of Protein
(Enhancer driven)
15
Tags Commonly Used in Mammalian Expression
Systems
16
Considerations in using mammalian
expression vectors
Type of promoter and enhancer sequence Type of
expression Amount of expression For
transfection of DNA into cells 1. Purity of
DNA 2. Method of DNA transfer
17
Common Promotor/Enhancer Elements
18
Common Mammalian Viral Expression Systems
19
Schematic of Recombinant Adenovirus system
Place gene of interest in viral
genome. Packaging into virion required. Can
use recombinant virus to infect cells for study.
20
Other Viral Vector Systems
21
Methods for Delivering DNA into Mammalian Cells

• Virus Mediated
• very efficient (approach 100)
• - Size limitation for virus packaging
• (2.5 kb SV40 6 kb retrovirus)
• - can get rearrangement during virus
• propagation.
• - cytopathic effect of virus limits protein
• expression time.
• Direct DNA Transfer
• - lower efficiency (5-50)
• less labor intensive then virus mediated

22
Methods of Direct Transfer of DNA into
Mammalian Cells
23
CaPO4 Precipitation
Factors influencing efficiency
pH Quality of DNA Growth State of Cells
Precipitates DNA onto cells which take up DNA by
endocytosis or phagocytosis
24
Liposomes
Relatively high efficency, useable on various
cell types, expensive and can be cytotoxic.
25
Electroporation can be used on all cell types,
needs to be optimized
for cell types and cost of machine. Microinjecti
on Used primarily for inserting DNA into
oocytes requires a
lot of skill. Viral Systems Can achieve 100
transfection Broad
host range Use in gene
therapy possible health
considerations.
26
Transient Expression
Short term (12-72hrs) high expression of
transgene Usually results in inhibition of cell
growth or result in the destruction of the
vector. Used for metabolic studies using a
reporter gene
27
Stable Expression Schemes

• Stable gene expression can be obtained by using
  vectors that
• integrate into the genome
• exist as episomes

28
Factors Affecting Transgene Expression
Number of copies of Transgene in cell Rate of
Transcription mRNA stability Integration
position
29
Other Methods for Converting Nucleic Acids into
Proteins

• In vitro translation systems
• Rabbit reticulocyte
• better for large proteins
• contain exogenous methionine
• sensitive to salts, hemin, Ca2, alcohols and
• detergents

2. Wheat germ extract better for smaller
proteins no exogenous methionine cheaper than
rabbit reticulocyte prone to premature
termination of translation can alter ionic
concentrations for optimal translation
30
Schematic of In-vitro Translation
ambion web page
RNA generated in vitro utilizes T7, SP6 or T3 RNA
polymerase in a separate uncoupled reaction.
31
Coupled In-vitro TranscriptionTranslation RR
System
System supplemented with RNA polymerase
32
Less Used In vitro TranscriptionTranslation
System
E. coli based system useful if cross-reactivity
is a problem with endogenous eukaryotic proteins
in RR
33
Xenopus Oocytes
Not as commonly used, offers high capacity for
translocation. extracts give consistent
processing of mRNA and stability of membranes
allow means of isolating and verifying
translocation products.
cDNA
transcription polyadenylation
translation modification assembly sorting
cRNA
cRNA
mature protein function
34
Baculovirus Systems
Utilizes Autographica californica nuclear
polyhedrosis virus (AcNPV) Takes advantage of
viral machinery by deletion of polyhedron
protein and p 10 gene product uses this late
viral promotor for expression of
transgene. Initial work is time consuming with
construction of transgene vector and screening of
virus plaques for recombinants.
35
Baculovirus Systems
36
RNAi
37
Strategies That Express dsRNA Triggers
in Mammalian Cells
38
Uses for RNAi
39
Proteomics a Quick Overview
40
(No Transcript)