Table I
The values of independent variables (post induction temperature and dissolved oxygen) and the corresponding levels used in a central composite design (normalized in –2, –1, 0, 1 and 2).
| Variable | Level | ||||
|---|---|---|---|---|---|
| –2 | –1 | 0 | 1 | 2 | |
| A: Post induction temperature (T) (°C) | 20 | 24.25 | 28.5 | 32.75 | 37 |
| B: Dissolved oxygen (DO) (%) | 10 | 20 | 30 | 40 | 50 |
Table II
The experimental design of 10 runs with two variables (post induction temperature and dissolved oxygen) and two responses (recombinant protein expression level and cell growth measured by absorbance at 600 nm).
| Run | Post induction Temperature (T) (°C) | Dissolved Oxygen (DO) (%) | Response 1 Recombinant Protein Expression Level (%) | Response 2 Cell Growth (Abs 600nm) | ||
|---|---|---|---|---|---|---|
| 1 | –1 | 24.25 | –1 | 20 | 8.0 ± 0.9 | 1.83 ± 0.18 |
| 2 | +1 | 32.75 | –1 | 20 | 15.3 ± 1.2 | 2.60 ± 0.19 |
| 3 | –1 | 24.25 | +1 | 40 | 10.1 ± 1.0 | 1.02 ± 0.11 |
| 4 | +1 | 32.75 | +1 | 40 | 8.4 ± 0.8 | 5.60 ± 0.29 |
| 5 | –2 | 20.0 | 0 | 30 | 4.6 ± 0.4 | 1.36 ± 0.11 |
| 6 | +2 | 37.0 | 0 | 30 | 11.1 ± 1.1 | 3.40 ± 0.22 |
| 7 | 0 | 28.5 | –2 | 10 | 10.0 ± 0.9 | 1.87 ± 0.21 |
| 8 | 0 | 28.5 | +2 | 50 | 3.8 ± 0.4 | 2.05 ± 0.20 |
| 9 | 0 | 28.5 | 0 | 30 | 14.6 ± 1.2 | 2.45 ± 0.19 |
| 10 | 0 | 28.5 | 0 | 30 | 14.2 ± 1.2 | 2.60 ± 0.19 |
Fig. 1.
The recombinant protein expression level analysis in different post induction times. (A) Western blot analysis of the proteins extracted from recombinant bacteria carrying pET39::β-NGF plasmid using anti-histag. HRP antibody. (B) % recombinant protein expression level measured by ImageJ software. All experiments were repeated three times under the identical experimental conditions.
Fig. 2.
The recombinant protein expression level analysis in different % DO and post induction temperature. (A) Western blot analysis of the proteins extracted from recombinant bacteria carrying pET39::β-NGF plasmid using anti-histag.HRP antibody. (B) % recombinant protein expression level measured by ImageJ software and comparison with the cell growth measured by absorbance at 600 nm. All experiments were repeated three times under the identical experimental conditions.
Table III
Parameter estimates and analysis of variance (ANOVA) of the model for recombinant production of β-NGF in E. coli and for cell growth.
| Source of variation | Degree of freedom | Sum of squares | Mean squares | F-value | p value (Probe > F) | |
|---|---|---|---|---|---|---|
| Recombinant Protein Expression Level (%) | Model | 5 | 141.50 | 28.30 | 120.36 | 0.0002 |
| A-Temperature (°C) | 1 | 29.14 | 29.14 | 123.94 | 0.0004 | |
| B-Dissolved Oxygen (%) | 1 | 24.65 | 24.65 | 104.85 | 0.0005 | |
| AB | 1 | 20.25 | 20.25 | 86.13 | 0.0007 | |
| A2 | 1 | 42.94 | 42.94 | 182.62 | 0.0002 | |
| B2 | 1 | 56.17 | 56.17 | 238.90 | 0.0001 | |
| Error | 1 | 0.058 | 0.058 | – | – | |
| Total | 9 | 142.44 | – | – | – | |
| Cell Growth (Abs 600 nm) | Model | 3 | 11.58 | 3.86 | 6.82 | 0.0232 |
| A-Temperature (°C) | 1 | 7.41 | 7.41 | 13.10 | 0.0111 | |
| B-Dissolved Oxygen (%) | 1 | 0.54 | 0.54 | 0.96 | 0.3655 | |
| AB | 1 | 3.63 | 3.63 | 6.41 | 0.0445 | |
| Error | 1 | 0.011 | 0.011 | – | – | |
| Total | 9 | 14.98 | – | – | – |
Fig. 3.
Normal (%) probability plot of the studentized residuals for the model of recombinant β-NGF production (A) and cell growth (B).
Fig. 4.
Response surface plots and their counter plots show the effect of post induction temperature and % dissolved oxygen on the protein (β-NGF) expression level (A and B) and cell growth (C and D).