This document describes site-directed mutagenesis experiments performed on beta-2 microglobulin (β2m) to study its role in amyloidosis. Primers were designed to introduce mutations D39V and I5T into β2m. Polymerase chain reaction and transformation were used to generate mutant plasmids, which were sequenced to confirm the mutations. Future studies are planned to introduce mutations W61F and W96F to further study β2m stability and aggregation.

1. SITE-DIRECTED MUTAGENESIS OF
Β2-MICROGLOBULIN
Tyler Liang, Austin College Welch Summer Research
Program

2. BETA2-MICROGLOBULIN PROFILE
 99 residue globular
protein
 subunit of the major
histocompatibility
complex I (MHC I)
 Immunity
 Filtered by glomerulus,
catabolized by proximal
tubular cells in the
kidney
Figure from Public database

3. THE “WHY?”
 Βeta2-Microglobulin
unfolds and aggregates
into amyloid fibrils
amyloidosis
 Accumulates in
synovial membranes
and osteoarticular sites
 Destructive
osteoarthropathies,
Carpal tunnel
syndrome,
tenosynovitis
http://www.jamesdisabilitylaw.com/images/Synovial_Membrane.gif
http://www.richmondchiro.net/wp-content/uploads/2010/04/carpal-tunnel-syndrome.jpg

4. THE “WHY?”
 Dialysis-related amyloidosis due to:
1.an increased serum concentration of β2m
(up to 60-fold) due to decreased renal
function
2. We need to find out…

5. PURPOSE OF MUTAGENESIS
 Comparison between
mutant and native
protein and pinpoint
differences
 Measure the
difference in free
energy, ΔG, between
mutant and native β2-
Microglobulin
Jahn, T.R. and S.E. Radford, The Ying and Yang of protein
folding. FEBS J, 2005. 272: p.5962-70

6. PRIMER DESIGN
D39V Revertent --- Length=45 Tm= 81.7 °C
Forward:
5’-CAT CCA TCC GAC ATT GAA GTT GAC TTA CTG AAG AAT GGA GAG AGA-3’
Complement:
3’-GTA GGT AGG CTG TAA CTT CAA CTG AAT GAC TTC TTA CCT CTC TCT-5’
Reverse Complement:
5’-TCT CTC TCC ATT CTT CAG TAA GTC AAC TTC AAT GTC GGA TGG ATG-3’
- 25 < x < 45 bases in length
- Tm ≥ 78 C
- Desired mutation should be in middle with ~ 10 – 15
bases of correct sequence on each side

7. PRIMER DESIGN
 Tm=81.5 + 0.41(%GC) –
675/N - % mismatch
- N is the primer length in
bases
- Values for %GC and
%mismatch are whole
numbers

8. METHOD: SITE-DIRECTED MUTAGENESIS AND
POLYMERASE CHAIN REACTION
 Reaction Ingredients:
5 uL - reaction buffer,
5 uL - dsDNA template,
1.25 uL - primer DNA,
1.25 uL - reverse primer DNA,
1uL - dNTP mix,
35.5 uL - DI water,
1 uL - Pfu Turbo DNA polymerase

9. METHOD: SITE-DIRECTED MUTAGENESIS AND
POLYMERASE CHAIN REACTION

10. METHOD: SITE-DIRECTED MUTAGENESIS AND
POLYMERASE CHAIN REACTION
20 cycles
95 C 1 min
60 C 30 sec
70 C 12 min Strategene. QuikChange Site-Directed Mutagenesis Kit Instruction
Manual. p.1-13

11. METHOD: DNA PURIFICATION
 PCR mix was then
centrifuged through filter to
bind the DNA to the silica
filter
 Water was then centrifuged
through to collect the DNA
 Agarose gel ran to confirm
success of PCR reaction
 DPN I was then added to
dispose of parent DNA

12. METHOD: AGAROSE GEL

13. METHOD: TRANSFORMATION
 XLI-Blue Super-competent cells: E. Coli. Cells
optimized and modified to be proficient at
transformation and plasmid synthesis
 pET29A: plasmid containing β2-Microglobulin gene
and Kanamycin resistance gene
Procedure:
 2 test tubes: 1. XLI-Blue with pET29A 2. XLI-Blue
with sterilized water
 Transformation induced by heat shock

14. METHOD: TRANSFORMATION
 Plated according to below table
Experimental Positive
Control
Negative
Control
Inoculation
Solution
XLI-Blue Cells
pET29A
XLI-Blue Cells
Sterile Water
XLI-Blue Cells
Sterile Water
Antibiotic Kan+ None Kan+
Expected Individual
colonies
Lawn of
bacterial growth
No growth

15. POSITIVE CONTROL

16. NEGATIVE CONTROL

17. EXPERIMENTAL

18. METHOD: GROWTH OF TRANSFORMED XLI-
BLUE CELLS
 5 tubes were prepared
with 7 mL, Kan+ treated LB
broth
 Colonies were then
individually chosen and
used to inoculate
respective growth tubes
 Cells were left in shaking
incubator at 37 C at 250
RPM for 20-24 hours

19. METHOD: EXTRACTION OF PET29A*
(MINIPREP)
 Lysis solution: lyse XLI-Blue
Cells
 Alkaline Protease enzyme:
denature endonuclease
enzymes
 Mix centrifuged through a
filter to bind DNA to silica
membrane filter
 Nuclease free water
centrifuged through same
filter to unbind and collect
the mutated plasmid

20. METHOD: DETERMINATION OF
CONCENTRATION OF DNA
 UV/Vis
Spectrophotometer used:
(370 nm – 220nm)
 Concentration of dsDNA
(C) = A260/.020

21. METHOD: SENDING IN THE SAMPLES
 50ng/uL
 10 uL samples
 TY1, TY2, TY3

22. PICTURE OF SEQUENCES

23. RESULTS
I5T TY6 7/3/12
ATG ATC CAG CGT ACT CCA AAG ATT CAG GTT TAC TCA CGT CAT CCA GCA GAG AAT GGA
ATG ATC CAG CGT ACT CCA AAG ATT CAG GTT TAC TCA CGT CAT CCA GCA GAG AAT GGA
AAG TCA AAT TTC CTG AAT TGC TAT GTG TCT GGG TTT CAT CCA TCC GAC ATT GAA GTT
AAG TCA AAT TTC CTG AAT TGC TAT GTG TCT GGG TTT CAT CCA TCC GAC ATT GAA GTT
GAC TTA CTG AAG AAT GGA GAG AGA ATT GAA AAA GTG GAG CAT TCA GAC TTG TCT TTC
GAC TTA CTG AAG AAT GGA GAG AGA ATT GAA AAA GTG GAG CAT TCA GAC TTG TCT TTC
AGC AAG GAC TGG TCT TTC TAT CTC TTG TAC TAC ACT GAA TTC ACC CCC ACT GAA AAA
AGC AAG GAC TGG TCT TTC TAT CTC TTG TAC TAC ACT GAA TTC ACC CCC ACT GAA AAA
GAT GAG TAT GCC TGC CGT GTG AAC CAT GTG ACT TTG TCA CAG CCC AAG ATA GTT AAG
GAT GAG TAT GCC TGC CGT GTG AAC CAT GTG ACT TTG TCA CAG CCC AAG ATA GTT AAG
TGG GAT CGA GAC ATG
TGG GAT CGA GAC ATG
TGGAGGCGGGTACATTCCCCTCTAGAATAATTTTGTTTAACTTTAAGAAGGAGATATGCAT..ATG ATC CAG
CGT ACT CCA AAG ATT CAG GTT TAC TCA CGT CAT CCA GCA GAG AAT GGA AAG TCA AAT
TTC CTG AAT TGC TAT GTG TCT GGG TTT CAT CCA TCC GAC ATT GAA GTT GAC TTA CTG
AAG AAT GGA GAG AGA ATT GAA AAA GTG GAG CAT TCA GAC TTG TCT TTC AGC AAG GAC
TGG TCT TTC TAT CTC TTG TAC TAC ACT GAA TTC ACC CCC ACT GAA AAA GAT GAG TAT
GCC TGC CGT GTG AAC CAT GTG ACT TTG TCA CAG CCC AAG ATA GTT AAG TGG GAT CGA
GAC ATG TAA TAA
GGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTG
CTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCT
AAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGC
GCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTT
CGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGT
TCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCC
TGATAGACGGTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGATCTTGTTCCAACTGGAACAACCCT
CAACCCTATTTCGGCCATTCTTTGATTTAAAGGGATTTGCCAATTCGGCCATTGGTAAAAATGGCTGATTACCAAAT
TTCCCGAATTTACCAAATTAACCTTAAATTAGGGGGACTTTCGGGAAATGGCGGAACCCATTGTTATTTTCAACTTC
AATGTTCCCTTGAATTTTTGAACCTCGGCCAAGAACGGTTTCCCGGATTACACTTTGAGCCCTTGAGGGACCCGGGC
TTGGGAGCGTGGGGTTGCACCAATATTCCTTAAGGAAAACCG
D39V and I5T
mutations were
successful

24. FUTURE STUDIES
 Mutagenesis for Tryptophan
mutations W61F and W96F
 Tryptophan 61: conformational
flexibility involving Trp61 for
binding the β2M subunit to the
MHC I complex
- increases tendency for self
aggregation
 Tryptophan 96: deep in
hydrophobic core; reports on
stability
http://biopsychiatry.com/tryptophan/tryptophan.jpg

25. ACKNOWLEDGEMENT
 Dr. John M. Richardson
 Arthur Yang, Laylee Ghafar
 Austin College Chemistry Department
 Robert A. Welch Foundation
 Austin College Cullen Fund
 Marian Cox Chemistry Fund

26. WORKS CITED
1. Basu A. Dialysis-Related Beta-2m Amyloidosis. MED REF. 2012.
http://emedicine.medscape.com/article/246542-overview
2. Jahn, T.R. and S.E. Radford, The Ying and Yang of protein folding. FEBS J,
2005. 272: p.5962-70
3. Raimondi S., et al., The two tryptophans of β2-microglobulin have distinct
roles in function and folding and might represent two independent responses
to evolutionary pressure. BMC EVOL BIOL, 2011. 11:159
4. Strategene. QuikChange Site-Directed Mutagenesis Kit Instruction Manual.
p.1-13
5. Vitiello A., Potter T.A., Sherman L.A, The Role of β2-Microglobulin in Peptide
Binding by Class I Molecules. SCIENCE, 1990. 250: p.1423-26
6. White H.E., et al., Globular Tetramers of β2-Microglobulin Assemble into
Elaborate Amyloid Fibrils. J MOL BIOL, 2009. 389(1): p.48-57