site directed mutagenesis of β2-microglobulin powerpoint presentation
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SITE-DIRECTED MUTAGENESIS OF Β2-MICROGLOBULINTyler Liang, Austin College Welch Summer Research Program
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
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
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…
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
PRIMER DESIGND39V 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
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
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
METHOD: SITE-DIRECTED MUTAGENESIS AND POLYMERASE CHAIN REACTION
METHOD: SITE-DIRECTED MUTAGENESIS AND POLYMERASE CHAIN REACTION
20 cycles95°C 1 min60°C 30 sec70°C 12 min Strategene. QuikChange Site-Directed Mutagenesis Kit
Instruction Manual. p.1-13
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
METHOD: AGAROSE GEL
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
METHOD: TRANSFORMATION
Plated according to below table
Experimental
Positive Control
Negative Control
Inoculation Solution
XLI-Blue CellspET29A
XLI-Blue CellsSterile Water
XLI-Blue CellsSterile Water
Antibiotic Kan+ None Kan+
Expected Individual colonies
Lawn of bacterial growth
No growth
POSITIVE CONTROL
NEGATIVE CONTROL
EXPERIMENTAL
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
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
METHOD: DETERMINATION OF CONCENTRATION OF DNA
UV/Vis Spectrophotometer used: (370 nm – 220nm)
Concentration of dsDNA (C) = A260/.020
METHOD: SENDING IN THE SAMPLES
50ng/uL 10 uL samples TY1, TY2, TY3
PICTURE OF SEQUENCES
RESULTSI5T 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 GGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGATCTTGTTCCAACTGGAACAACCCTCAACCCTATTTCGGCCATTCTTTGATTTAAAGGGATTTGCCAATTCGGCCATTGGTAAAAATGGCTGATTACCAAATTTCCCGAATTTACCAAATTAACCTTAAATTAGGGGGACTTTCGGGAAATGGCGGAACCCATTGTTATTTTCAACTTCAATGTTCCCTTGAATTTTTGAACCTCGGCCAAGAACGGTTTCCCGGATTACACTTTGAGCCCTTGAGGGACCCGGGCTTGGGAGCGTGGGGTTGCACCAATATTCCTTAAGGAAAACCG
D39V and I5T mutations were successful
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
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
WORKS CITED1. 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
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