Background
Materials and methods
Materials
Mutagenesis design and method
Amino acid | Codon ( E. coli) | Amino acid | Codon ( E. coli) |
---|---|---|---|
Phe/F | TTT | Leu/L | CTG |
Tyr/Y | TAT | Pro/P | CCG |
Try/W | TGG | Gln/Q | CAG |
Ile/I | ATC | Val/V | GTT |
Ser/S | AGC | Ala/A | GCG |
Met/M | ATG | Asp/D | GAT |
Thr/T | ACC | Glu/E | GAA |
Asn/N | AAC | Gly/G | GGT |
Inclusion body expression, purification, and refolding screening
Activation and kinetic measurements
Fibrinolysis assay
Staphylokinase (SAK) expression and purification
Results
mPlm | Active μM in 0.5 μM | Vmax (μM/Min) | Km (μM) | Kcat (Min-1) | Kcat/Km | Kcat/Km Ratio |
---|---|---|---|---|---|---|
(μM-1Min-1) | ||||||
01_K698P | 0.00375 | 11 ± 0.5 | 2145.3 ± 145.5 | 2933 | 1.37 | 0.29 |
02_WT
|
0.1265
|
207 ± 4.9
|
347 ± 25
|
1636
|
4.72
|
1
|
03_K698L | 0.0685 | 126 ± 6.8 | 2459 ± 201 | 1839 | 0.75 | 0.16 |
04_K698F
|
0.124
|
97 ± 4.6
|
439 ± 59
|
782
|
1.78
|
0.38
|
05_K698D | 0.046 | 17 ± 1 | 587 ± 83 | 370 | 0.63 | 0.13 |
06_K698W | 0.0065 | 72 ± 8 | 9686 ± 1222 | 11077 | 1.14 | 0.24 |
07_K698T | 0.007 | 24 ± 1 | 4107 ± 221 | 3429 | 0.83 | 0.18 |
08_K698N
|
0.1625
|
224 ± 4.4
|
811 ± 35
|
1378
|
1.7
|
0.36
|
09_K698I | 0.399 | 152 ± 6.6 | 1587 ± 118 | 381 | 0.24 | 0.05 |
10_K698Q
|
0.129
|
213 ± 6.5
|
1406 ± 76
|
1651
|
1.17
|
0.25
|
11_K698E | 0.1905 | 62 ± 2.8 | 2411 ± 162 | 325 | 0.13 | 0.03 |
12_K698V
|
0.154
|
202 ± 5.6
|
1609 ± 77
|
1312
|
0.82
|
0.17
|
13_K698G | 0.108 | 11 ± 1.4 | 617 ± 181 | 102 | 0.17 | 0.03 |
14_K698M
|
0.127
|
221 ± 5.6
|
1664 ± 71
|
1740
|
1.05
|
0.22
|
15_K698Y | 0.3545 | 33 ± 1.9 | 2451 ± 209 | 93 | 0.04 | 0.01 |
16_K698S | 0.1405 | 39 ± 1.3 | 1776 ± 96 | 278 | 0.16 | 0.03 |
17_K698A | 0.046 | 11 ± 0.7 | 885 ± 111 | 239 | 0.27 | 0.06 |
conc. (mg/mL) | Digestion area (mm2)* | ||
---|---|---|---|
WT | K698N | K698Q | |
0.05 | 125.7 ± 4.2 | 126.7 ± 8.5 | 71.1 ± 10.6 |
0.1 | 194.9 ± 8.8 | 151.8 ± 9.3 | 100.8 ± 20.1 |
0.2 | 217.9 ± 16.6 | 189.9 ± 1.7 | 138.0 ± 13.2 |
0.4 | 279.1 ± 2.1 | 233.8 ± 9.6 | 182.9 ± 18.6 |
0.8 | 308.1 ± 22.0 | 268.8 ± 4.1 | 203.7 ± 10.7 |