Ld PEX5-Qfl-M123-H6
DNA Sequence
523 ATGAACCTAG CACCAGCGGC GCAGGACTCG GCGTGGGCGG ACCAGCTCAG CCAGCAACAA 582
583 TGGAGCACCG ACTACTCGCA GGTGCAGACC TTCAGTGCAC CAGGGATGGA GGACAAGACG 642
c c gc
643 GTGGAGGAGC GGATAAAGGA TAGCGAATTC TACAAGTTCA TGGACCAGGT CAAGAATAAG 702
c c g c
703 GAGGTACTGA TTGACGAGGA GAAGGGCGAG CTGGTGCAGG GCCCCGGCCC CGAGGTCGGG 762
c c c gc c
763 GTGCCAGAGG ATGCGGAGTA CCTTCGTCAC TGGGCCGAGA TGGAGGGGCT ACACATGCCA 822
823 GAGAGCGTCT TTCAGTCGCC TCCACCGGCG AGCGCGATGA CGTCTCCGGA GAACGGGGAC 882
883 CCGGACGCGT ACGTCAAGGA GATGGATATG GCCGCGAACG ACGTCGAGGA CTGGGCGCAG 942
943 GAGTATGCGG AGATGCAGGA ACGCCTGCAG AAGGTGACGA ACAGCACCGA CTACCCCTTC 1002
1003 GAGCCTAACA ACCCGTACAT GTTCCACGAC TTTCCGTTCG ATGAGGGGAT GGAGATGCTC 1062
1063 CAGCTTGGCA ACCTTGCCGA GGCCGCTCTT GCTTTTGAGG CGGTTTGCCA TAAGGACAGC 1122
1123 AGTAATGAGA AGGCATGGCA GATTCTTGGA ACGACGCAGG CGGAGAACGA GAAGGACGGG 1182
1183 CTGGCCATCA TTGCGCTCAA CAACGCCCGC AAACTGAACC CCCGCAACCT CGAGGTTCAC 1242
1243 GCCGCGCTGA GCGTGTCGCA CACGAACGAG CGCAACGCCG ACGCCGCCAT GGACTCGCTG 1302
1303 AAGGCGTGGC TCATAAATCA TCCCGAGTAT GAGCAGCTCG CCTCCGTGTC TATCCCGCCT 1362
1363 AACGCGGAGC TCGACGTGCA GGAAACGTTC TTCTTTGCCG ATCCGTCCCG CATGCGGGAG 1422
1423 GCCCGTACCC TCTATGAAGC CGCCATCGAG ATGAACCCGA GCGACTCGCA GCTTTTCACG 1482
1483 AATCTCGGCG TGCTGCACAA TGTGGCACAT GAGTTCGACG AGGCTGCCGA GTGCTTCCGC 1542
1543 AAGGCGGTCG CGCTGCACCC CGATGACCCC AAGATGTGGA ACAAGCTCGG CGCCACCCTG 1602
1603 GCAAACGGCG GCCACCCTGA CCAGGCACTG GAGGCGTACA ATCGCGCTCT GGACATCAAC 1662
1663 CCAGGCTATG TACGGGCCAT GTACAACATG GCAGTGGCGT ACAGCAACAT GTCGCAGTAC 1722
1723 AACATGGCCG CCCGCCAAAT TGTCAAGGCG ATCGCGTCGC AGCAGGGCGG CACGAAGCCC 1782
1783 AGCGGCGAGG GTTCCATCAT GGCGACACGC AACATGTGGG ACCTTCTGCG TATGACGCTG 1842
1843 AACCTCATGG ACCGCGATGA CTTGGTGCAG CTGACATACA ACGAGCAATT GGAGCCGTTT 1902
c gc c c
1903 GTGAAGGAGT TCGGACTTGA GGGCCACGTC GCGGCCGCAC TCGAGCACCA CCACCACCAC 1962
1963 CACTGA 1968
The underlined sequence in blue is from the vector.
The sequence in red is mutated.
Vector = pET29b
Resistance = kanamycin
Protein Sequence
175 MNLAPAAQDS AWADQLSQQQ WSTDYSQVQT FSAPGMAAAT VAAAIKDSEF YKFMDQVKNK 234
235 EVLIAAAAGA LVQGPGPEVG VPEDAEYLRH WAEMEGLHMP ESVFQSPPPA SAMTSPENGD 294
295 PDAYVKEMDM AANDVEDWAQ EYAEMQERLQ KVTNSTDYPF EPNNPYMFHD FPFDEGMEML 354
355 QLGNLAEAAL AFEAVCHKDS SNEKAWQILG TTQAENEKDG LAIIALNNAR KLNPRNLEVH 414
415 AALSVSHTNE RNADAAMDSL KAWLINHPEY EQLASVSIPP NAELDVQETF FFADPSRMRE 474
475 ARTLYEAAIE MNPSDSQLFT NLGVLHNVAH EFDEAAECFR KAVALHPDDP KMWNKLGATL 534
535 ANGGHPDQAL EAYNRALDIN PGYVRAMYNM AVAYSNMSQY NMAARQIVKA IASQQGGTKP 594
595 SGEGSIMATR NMWDLLRMTL NLMAAAALVQ LTYNEQLEPF VKEFGLEGHV AAALEHHHHH 654
655 H 655
Number of Residues = 481
MW of S-Met Protein = 53214.44 Da
MW of SeMet Protein = 54339.80 Da
Theoretical pI = 4.70
Extinction Coefficient e280 (ml/mg.cm) = 1.247
[Divide A280 by e280 to obtain conc. in mg/ml]
Concentration Conversion:
1 mg/ml = 0.01879 mM, 1 mM = 53.21 mg/ml
Number of positively charged residues (R + K) = 31
Number of negatively charged residues (D + E) = 68
Net Charge = -37
Number of hydrophilic residues = 242 (50.31 %)
Number of hydrophobic residues = 239 (49.69 %)
Amino Acid Count %
A (Ala) 73 15.2
E (Glu) 41 8.5
L (Leu) 39 8.1
N (Asn) 31 6.4
D (Asp) 27 5.6
P (Pro) 27 5.6
S (Ser) 26 5.4
Q (Gln) 26 5.4
V (Val) 25 5.2
M (Met) 24 5.0
G (Gly) 22 4.6
H (His) 18 3.7
T (Thr) 17 3.5
K (Lys) 17 3.5
F (Phe) 17 3.5
Y (Tyr) 15 3.1
R (Arg) 14 2.9
I (Ile) 12 2.5
W (Trp) 8 1.7
C (Cys) 2 0.4
Atomic composition:
Carbon C 2338
Hydrogen H 3565
Nitrogen N 641
Oxygen O 733
Sulfur S 26
Formula: C2338H3565N641O733S26
Total number of atoms: 7303
Extinction coefficients:
Conditions: 6.0 M guanidium hydrochloride
0.02 M phosphate buffer
pH 6.5
Extinction coefficients are in units of M-1 cm-1 .
The first table lists values computed assuming ALL Cys
residues appear as half cystines, whereas the second table
assumes that NONE do.
276 278 279 280 282
nm nm nm nm nm
Ext. coefficient 65095 65927 65575 64840 62920
Abs 0.1% (=1 g/l) 1.223 1.239 1.232 1.218 1.182
276 278 279 280 282
nm nm nm nm nm
Ext. coefficient 64950 65800 65455 64720 62800
Abs 0.1% (=1 g/l) 1.221 1.237 1.230 1.216 1.180
Estimated half-life:
The N-terminal of the sequence considered is M (Met).
The estimated half-life is: 30 hours (mammalian reticulocytes, in vitro).
>20 hours (yeast, in vivo).
>10 hours (Escherichia coli, in vivo).
Instability index:
The instability index (II) is computed to be 39.63
This classifies the protein as stable.
Aliphatic index: 71.60
Grand average of hydropathicity (GRAVY): -0.414
Expression (1 Liter culture)
- Grow ON 10 ml of culture with a single colony in LB + 0.5 ug/ml kanamycin.
- Inoculate 1 liter of LB (+ kanamycin) with the ON culture.
- Grow at 37o C till OD600 ~ 0.5
- Induce with 1 mM IPTG and express for 3-4 hours.
- Harvest the cells.
Purification
[PN7= 20 mM Phosphate (pH 7.0) + 0.5 M NaCl ]
- Resuspend the cells in PN7 + 1 mM PMSF (or Protease Inhibitor cocktail tablet).
- French-press twice.
- Centrifuge and save supernatant.
- Ni-Col: Resuspend the pellet in PN7 + 1 mM PMSF, bind to Ni-col,
elute with Imidazole. (Protein elutes between 20-100 mM Imidazole conc.)
- Dialyze against 20 mM Tris (pH 7.0) + 50 mM NaCl + 1 mM EDTA/PMSF.
- Anion Exchange: Load the sample on AE, elute between 0-1 M NaCl
gradient. (Protein elutes between 25-40 mS/cm conductivity.)
- Size Exclusion OR Concentration OR Dialysis in a 50 kDa Mol Wt. Cutoff
membrane: use 20 mM Tris (pH 7.0) + 100 mM NaCl as running buffer.
- Dialyze to 20 mM Tris (pH 7.0) and concentrate to > 10 mg/ml.