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