Optimization of Secreted Recombinant Human Epidermal Growth Factor Production Using Pectate Lyase B from Escherichia Coli BL21(DE3) by Central Composite Design and Its Production in High Cell Density Culture.

CONTEXT: Human Epidermal Growth Factor (hEGF) is a potential therapeutic protein that has been widely used as a healing agent for various chronic wounds. It induces the proliferation and metabolism of epithelial cells, regenerates skin cells, and validates skin elasticity. In the previous study, recombinant hEGF (rhEGF) had been successfully expressed extracellularly in Escherichia coli (E. coli) BL21 (DE3) using pectate lyase B (PelB) signal peptide. The previous study has shown that the medium concentration and the induction time influenced the production of rhEGF. AIMS: Therefore, this study was conducted to optimize the induction time and medium concentration for rhEGF extracellular secretion then followed by scale-up production. SETTINGS AND
DESIGN: This experiment was carried out using E. coli BL21 (DE3) which contains pD881 plasmid that carries hEGF and PelB gene. Optimization design of induction time and medium concentration were obtained using Central Composite Design (CCD). METHODS AND MATERIAL: The method of study started by the rejuvenation of E. coli culture, extracellular secretion, and optimization in the flask scale then followed by scaled-up production with high-cell density culture in the fermenter. STATISTICAL ANALYSIS USED: The optimization was carried out using Response Surface Methodology (RSM) and multi regression analysis.
RESULTS: This work showed that the multiplication of 1.5-fold medium concentration with induction time 3h after the culture started gave the best result among another condition in this study. Additionally, the rhEGF production in the fermenter scale was identified by SDS-PAGE Tricine and quantified by ELISA, which showed 122.40 μg of the rhEGF per milliliter medium.
CONCLUSIONS: In respect of the result, we conclude that the optimized condition of extracellular secretion was successfully obtained, and gives higher result before the previous study. Copyright:

Introduction

In both science and engineering, determining the factors that significantly affect the system response is often difficult. Usually full factorial design of the experiment is used to test all possible combinations of various factors was did not effective. Furthermore, the interpretation of a large set of measurement data is difficult and may be unnecessary, because especially in engineering and practical applications, focus on only trends in how factors affect system response can be sufficient. Therefore, response surface methodology combined with central composite design (CCD) was choosen.[1]

Human epidermal growth factor (hEGF) is a single-chain polypeptide composed of 53 amino acid residues, with six cysteine residues forming three disulfide bonds, which help fold the hEGF protein precisely, thus, it can be biologically active.[28] The hEGF protein weighs 6.2kDa is synthesized by the hEGF-coding gene located on the 4q25-q27 chromosome, which has unique characteristics, that is, thermal resistance and is not denatured by heat up to 70°C.[3] Moreover, hEGF is a growth factor that induces the proliferation and the metabolism of epithelial cells and has been widely used as a healing agent of various chronic wounds, and also as cosmetics to regenerate skin cells and validate skin elasticity.[4]

Recombinant proteins produced by the Escherichia coli (E. coli) host have several advantages including faster expression processes.[5] It is because of their ability to overgrow, relatively inexpensive for the production of recombinant proteins compared to other expression systems due to its higher density on an inexpensive substrate. Moreover, its genetic has been well-characterized and the availability of a large number of mutant host strains that have certain advantages.[6]

Expression of the hEGF recombinant (hEGF) gene with the pectate lyase B(PelB) signal peptide in E. coli BL21(DE3) secreted hEGF through periplasm to the medium. Optimized culture condition may influence the successful production of recombinant protein, not only the success of the gene insertion in the host microorganism solely.[78] In this study, medium concentration and induction time were optimized in producing rhEGF in flask scale based on central composite design. The optimization was then followed by high cell density culture to scale-up the production of hEGF in 2 L-working volume fermentors using the optimized condition based on flask scale result.

Materials and Methods

Expression optimization using the central composite design

In the experimental design of the central composite, the three-level variables, that is, minimum (−1), medium (0), and maximum (1), were determined for both medium concentration and induction time [Table 1]. These three variables were then inserted into Minitab[1] statistical software (Trial version) to generate the experimental design. Afterward, a total of 1 mL starter culture of E. coli BL21(DE3) (pD881-PelB, synthesized in Atum, California) was introduced into 100 mL Luria Bertani (LB) medium containing kanamycin (Sigma-Aldrich) in 500 mL shaken flask at 37°C with 200rpm shaking rate. L-Rhamnose (Sigma-Aldrich) with optimum concentration 4mM was used as an inducer and added into the medium for certain hours (as showed in Table 1) after the culture started.

Table 1

Factor and level central composite design

Factors	Level
	−1	0	1
Medium concentration (mM)	1	1.5	2
Induction time (hours)	3	6	9

Production human epidermal growth factor protein in the fermenter

The production of hEGF protein was carried out by aerobic fermentation E. coli BL21(DE3) containing plasmid of pD881-PelB-hEGF[7] in 10L fermenter (Major Science MS-F1) with working volume 2L. The fermentation process was performed in three phases, that is, (1) growth phase, this step was carried out by inserting 1% (vol/vol) inoculum into 2L (medium). Fermentation medium concentration was increased to 1.5-fold. The culture was set at 37°C, with shaking rate of 250rpm for 4h. The pH of the medium was maintained at 7.0 with the addition of 2 M sodium hydroxide (Merck) and 2 M sulfuric acid (Merck) set automatically; (2) The fed-batch phase, this phase was carried out for 3h by feeding the culture with medium containing 426g/L glycerol, 85g/L tripton, 43g/L yeast extract, and 5.3g/L magnesium sulfate heptahydrate; (3) Induction phase, this phase was performed by adding 2mM L-Rhamnose into the culture after 3h of feeding. The hEGF product collected in supernatant media after 20h induction time.

Protein characterization with SDS-PAGE tricine

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Tricine (Biorad Power PacTM Basic) was performed to identify the secreted rhEGF. A total of 20 μL of a mixture of sample and buffer (1:1 (vol/vol)) was loaded into the gel well. In this study, a total of 7 μL protein marker biorad (25kDa) was used as a marker of protein size. Electrophoresis was performed for 2h. The gel was electroporated in 30 V for 30min before the voltages were increased to 100 V and ran for 2h. The gel was then stained at 25°C in the dark for 18h. The gel was then washed by using distilled water, and the gel was incubated in the destaining solution for 6h at 25°C. The protein band (6.2kDa) was then captured.

Quantification of recombinant human epidermal growth factor with enzyme-linked immunosorbent assay (ELISA)

The quantification of rhEGF was performed by following the procedure as described in Quantikine ELISA KIT hEGF (R and D System). Absorbance measurement was carried out at λ450 nm with correction factors at λ540 nm, and λ570 nm.

Results and Discussion

Expression optimization with the central composite design

In this study, optimization of rhEGF production from E. coli BL21(DE3) (pD881-pelB) was performed following the experimental design generated by Minitab for CCD. The variable determined for medium concentration and induction time in CCD was adapted from the previous study.[7]

The lowest and the highest medium concentration was set into the design were 1 and 2mm, respectively [Table 1]. On the other hand, the lowest and highest induction time was set at 3 and 9h, respectively [Table 2]. Experiment in 500 mL shaken flask containing 100 mL culture was carried out based on the generated design. Afterward, the amount of rhEGF produced in the medium was measured and analyzed [Table 3].

Table 2

Experimental design based on medium concentration and induction time

Standard order	Run order	Point type	Parameters
			Medium concentration	Induction time
1	1	1	1.0	3
2	2	1	2.0	3
3	3	1	1.0	9
4	4	1	2.0	9
5	5	−1	1.0	6
6	6	−1	2.0	6
7	7	−1	1.5	3
8	8	−1	1.5	9
9	9	0	1.5	6
10	10	0	1.5	6
11	11	0	1.5	6
12	12	0	1.5	6
13	13	0	1.5	6

Table 3

Human epidermal growth factor content in fermenter sample

Dilution factor (× 106)	Absorbance	Human epidermal growth factor (ng m/L)
2	0.558	121.76
25	0.181	115.30
80	0.161	130.15

Based on the parameters tested, a mathematical equation was generated. The equation explains the relationship between the linear influence of each parameter and its interaction. The equation was written as follows:

y = –35.0 + 244.8 X1 - 23.2 X2 - 84,6 X11 + 0.925, X22 + 6.20 X12

On the basis of the above equation, y represents the amount of rhEGF as the response of both factors. X1 represents the medium concentration, and X2 represents the induction time. The coefficient value obtained describes a relationship to the level of protein achieved. A positive coefficient value for the density of medium indicates a positive correlation between the medium concentration and the protein content produced.[1] In contrast, a negative coefficient of induction time showed that the longer induction time could decrease the protein content produced. The effect of the variables on auto Trm activity was observed by the contour plot [Figure 1] and surface plot [Figure 2]. The plots showed that optimum condition to express rhEGF was in medium concentration 1.5-fold with 3h of induction time after the culture started. Additionally, by using the equation above, the produced protein content of the expression was predicted with 8.43 and 91.57% of the average percentage difference and the average accuracy, respectively.

Figure 1

Contour plot of protein level (ng m/L) in function of medium concentration and induction time (h)

Figure 2

Surface plot of protein level (ng m/L) in function of medium concentration and induction time (h)

Production human epidermal growth factor protein using a fermenter

The optimum condition generated from rhEGF production optimization in the shaken flask was applied in scaled up production in 10L fermenter with 2L working volume. The rhEGF output in fermenter underwent three phases. The first phase was the growth phase to grow the host E. coli BL21(DE3) (Pd881-PelB) until it reached sufficient density to produce rhEGF. The second phase was the feeding phase to increase the cell density and to prepare for the induction phase. Substances added into the feeding medium were supportive for large-scale expression that had been consumed in the growth phase. Finally, the third phase corresponds to the induction phase that was carried out 3h after feeding by adding L-Rhamnose 4mM into the medium.

Characterization of the rhEGF produced in fermenter scale can be visualized on SDS-PAGE Tricine electrophoreogram in Figure 3. Our result showed that rhEGF gene was successfully expressed from BL21(DE3) with a pD881-PelB expression vector. The bands support this result appeared below 10kDa, which is estimated as a rhEGF band (~6.2kDa).

Figure 3

SDS-PAGE Tricin electrophoregram of rhEGF expression from the medium in fermenter scale: (1) Marker, (2) The soluble fraction 8h after induction, (3) Medium fraction at induction, (4) medium fraction 8h after induction, (5,6,7) Medium fraction 20h after initiation

Quantification of recombinant human epidermal growth factor by ELISA

ELISA performed quantification of rhEGF expressed in the medium in fermenter scale. The hEGF standard was provided in the kit. Our result revealed that rhEGF expressed in the medium in the fermenter scale was 122.40 μg m/L [Table 3]. The result was 1000-fold higher than the shaken flask scale production. This result indicated that this strategy could be used to scale-up the production of rhEGF extracellularly from E. coli BL21(DE3) (Pd881-PelB).

Conclusion

The optimum condition of extracellular production of rhEGF from E. coli BL21(DE3) based on CCD result was a medium concentration of 1.5-fold and induction time 3h after the culture started. This optimum condition is successfully applied in the scaled up production of rhEGf in 2L working volume fermenters. The rhEGF produced from fermenter scale is 122.40 μg m/L on the 20h in the medium after induction.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.