Or:

1
Or Isolation of recombinant protein mutants with
altered binding properties
Why are they doing this? Problem Binding of TPA
to liver cells leads to clearance from the
bloodstream Want to avoid clearance in TPA
therapy (anti-thrombolytic, clot buster) MAb387
blocks binding to cultured hepatoma cells
(liver-like) MAb387 decreases clearance rate.
Goal Mutate the cloned TPA gene. Mutate it in
the MAb438-binding region ? mutant TPA that Is
hepatocyte binding-negative (select) Is still
protease (remains catalytically active) (screen)
2
How could one do this? Select? Need to
characterize many mutant proteins, and find the
protein with the desired characteristic, and then
rescue the gene for that protein. Mammalian
cell transfectant But TPA is secreted, so
protein becomes divorced from the DNA that coded
it. But Coffino and Scharff had a technique for
looking at secretory variants (of myeloma cells)
Immunoprecipitate secreted proteins around
colonies grown in agar (Ig secretion,
precipitation by anti-mouse-Ig antibody)
3
All in soft agar
Medium in agar
Imagine Antibody in top layer MAb387 Colonies
CHO cell permanent co-transfectants of mutant
library TPA
Longer. Colonies may not make enough. But you
dont need a FACS ()
Precipitate -
Precipitate
Coffino and Scharff (Proc Natl Acad Sci U S A.
1971 Jan68(1)219-23.)
4
Phage display a way to link the variant protein
to its coding DNA Mutagenize the gene as a fusion
protein to a phage coat protein and make a
library in bacteriophage. The mutants will be
displayed on the surface of the phage and can be
panned for (or against).
DNA
DNA
DNA
Here, on would collect the members of the phage
library of mutant TPAsthat dont stick to
hepatoma cells, or to immobilized MAb328. Lots of
noise no-stickers could be for whatever reason
(denatured, statistical, etc )
5
Authors here use a mammalian cells as the carrier
of the DNA and the cell surface as a display
site. Fusion protein to a membrane anchor
protein (peptide, really). What did they
do? Mutagenesis What region? 333 bp K1
(kringle-1), known to bind the MAb438, which
competes for hepatocyte binding (so same target
epitope). How did they get it mutated? Error-pron
e PCR How did they isolate just the kringle 1
region? PCR fragment. How did they get the
mutagenized fragment back in? Introduced
restriction sites at the ends, w/o affecting the
coding.
6
What did they put the mutagenized fragment
into? DAF fusion protein How did they get into
into cells? Electroporation, transient
fusion. How many copies per cell. And why is
that important? One, by electroporation at low
DNA concentration. In a transient
transfection! Binding is dominant. How did
they select cells making MAb438-non-binding
TPA? FACS
7
(No Transcript)
8
How did they recover the plasmid carrying the
mutant TPA gene from the selected cells? Hirt
extraction. Like a plasmid prep, high MW DNA
allowed to get entangled and form a
clot. Centrifuge. Chromosomal DNA ? soft
pellet plasmid DNA circles stay in supernatant.
Then re-transfect, re-sort in FACS. Make a new
library in secreted form (no DAF) for
testing Now clone E. coli colonies and test
individually for Hepatoma cell binding.
How?Label WT TPA with fluorescein (FITC)
(conjugate chemically) Mix with hepatoma cells
and analyze on a flow cytometer (FACS w/o the
sorter part). See specific and non-specific
binding. Subtract non-specific and the amount
not competed by excess un-labeled TPA
9
FACS Fluorescence-activated cell sorter
Impart a charge on the recognized cell
10
No. of cells
Having this much fluorescence
11
(No Transcript)
12
Analysis on 2 colors
One cell
Amount of green fluorescence (log)
Say, want high reds but low greens. Deflect these
Amount of red fluorescence (log)
13
MAb to protease domain
enriched
Low kringle-1 reactivity
MAb to kringle-1 domain
14
WT
Compete. So still bind.
15
(No Transcript)
16
(No Transcript)