High-level expression of soluble human epidermal growth factor in Escherichia coli

doi:10.21203/rs.3.rs-1646227/v1

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High-level expression of soluble human epidermal growth factor in Escherichia coli

https://doi.org/10.21203/rs.3.rs-1646227/v1

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Human epidermal growth factor (hEGF) has multiple biological functions, and it is a very expensive polypeptide with attractive market prospects. However, the production of hEGF needs for high cost to manufacture polypeptide demands reinvestigations of process conditions so as to enhance economic benefits. Improving the expression of soluble hEGF is the fundamental method to reduce the cost. In this study, a non-secretion engineered strain of biosynthesized hEGF was constructed, using plasmid pET-22b(+) in Escherichia coli. Preliminary fermentation and high cell density cultivation were carried out in shake flasks and in a 5 L bioreactor, respectively. A high yield of 98 ± 10 mg/L of soluble hEGF and a dry cell weight (DCW) of 6.98 ± 0.3 g/L were achieved in shake flasks. Then, fermentation conditions were optimized for large-scale production, while taking into consideration the expensive equipment required for cooling and conforming to industrial standards. A yield of 285 ± 10 mg/L of soluble hEGF, a final cell density of 57.4 ± 2 g/L DCW (OD₆₀₀ = 141.1 ± 4.9), and hEGF productivity of 14.3 mg/L/h were obtained using a bioreactor at 32 ℃ for 20 h. The production method developed in this study for the biosynthesis of soluble hEGF is efficient and inexpensive.

Escherichia coli

Epidermal growth factor

Protein expression

High cell density cultivation

Low-cost

Epidermal growth factor (EGF) is an important autocrine and paracrine signaling growth factor for humans (Cohen 1965; Cohen & Carpenter 1975; Le et al. 2009). Human epidermal growth factor (hEGF) was isolated from urine by Gregory’s team in 1975 and obtained in crystal form by Hideo Ogiso and co-workers (Gregory 1975; Ogiso et al. 2002). hEGF is a single-chain polypeptide consisting of 53 amino acid residues, wherein three pairs of disulfide bonds constitute the active center of hEGF (Cooke et al. 1987). Crucially, hEGF is known to promote cell proliferation, differentiation, and migration (Cohen 1965; Cohen & Carpenter 1975; Gregory 1975). Therefore, hEGF is of great importance in medicinal (Esquirol Caussa & Herrero Vila 2015) and cosmetic fields (Gauglitz et al. 2011). It needs to be batch synthesized because of its marked biological function (Yera-Alos et al. 2013) and its current market price of 1.05 million ($) per gram (Sigma offered). In addition, the expression of soluble polypeptides is usually not high, especially for proteins with a small number of amino acid residues, such as 51 amino acid insulin (Bhoria et al. 2022).

For this reason, many polypeptides of interest for the pharmaceutical and economic industry are prepared employing the DNA recombination technology (Baeshen et al. 2014; Liu et al. 2017; Jia et al. 2018). This is because chemical synthesis of polypeptides should not only maintain their chirality and activity, but also address concerns of pollution and damage to the environment. For example, insulin was the first protein to be entirely synthesized in more than 41 steps, using intricate chemical reactions of toxicant reagents, such as sulfonic acid and halic acid. Currently, the insulin and fibroblast growth factor have been manufactured via biotransformation using gene recombination operation in industry (Goeddel et al. 1979; Jang & Ahn 2019; Lin et al. 2019). Even though many of polypeptides have been industrialized, they are frequently low economic benefits due to their low yield in industry and expensive intricate process conditions, such as low-level soluble expression, low-temperature induced (below 20 ℃), and specific inducers usually required (Isopropyl-beta-D-thiogalactopyranoside).

This is the situation of polypeptides, hence, the process conditions need to be reconsidered based on decreased costs. The costs of the processes are increased mainly due to renaturation for inclusion bodies. These low molecular weight polypeptides are applied in medicine and cosmetic fields because of their biological activity. However, recombinant proteins, especially low molecule polypeptides, are synthesized in the cytoplasm through a non-secretory method, when the rate of synthesis is greater than the correct folding rate of the protein to form inclusion bodies, thereby reducing the production of the active target (Kane & Hartley 1988; Abdull Razis et al. 2008). In other words, the peptide chains after mRNA translation cannot spontaneously form the original spatial conformation of the protein, but the peptide chains folding occurs randomly, resulting in incorrect conformation. Additionally, refolding of inclusion bodies is a challenge, mainly because of low refolding rate and also as the refolding time and cost are higher than the separation and purification costs (Han et al. 2012; Singhvi et al. 2020). Reducing the formation of inclusion bodies will significantly reduce the cost of protein expression in non-secretory systems. Improving the expression of soluble hEGF is the fundamental method to reduce the cost. Therefore, industrial production of low-cost hEGF necessitates reasonable strategies to improve expression of soluble hEGF in order to increase the economic benefits.

Traditionally, three major norms have been used to optimize the biochemically process: high yield, high productivity, and high titer (Three high). Frequently, the researchers will reasonably choose one of them as a criteria of optimization process. Additionally, in order to obtain high yields or productivity or titer, the cost of inputs is huge. For instance, we mainly tested reducing the number of inclusion bodies decreasing the inducing temperature. The induced temperature in the shake flask can be reduced, but this strategy incurs additional costs of expensive cooling equipment and long cooling times in the industry. It was reported that raising the fermentation temperature by 5 ℃ can save $30000 in industrial cost, especially induced temperature (D'Amore et al. 1989). Briefly, obtained the one of ‘three high’ does not necessarily bring high economic benefits. We should pay attention to the importance of this concept in industrial application. The new generational principles of economic optimizations are to obtain high economic benefits, which an economic balance is judged from high yield, high productivity, and titer (Fig. 1). Their results could be decreased on account of this new generational principles, but a simple and easy to operate process and high economic results are obtained at minimum costs. There are several examples of the successful implementation of biotransformation using non-optimal process conditions for the manufacturing of drugs applied in medicine (Xu et al. 2015; Sun et al. 2019; Liu et al. 2022).

In this study, a non-secretory recombination Escherichia coli LS-1 strain with a strong T₇ promoter was constructed to express hEGF (Fig. 1). First, the expression of hEGF in the shake flask was evaluated. Then, high cell density cultivation (HCDC) in a complex medium was carried out to investigate characteristics of the fermentation process and to improve the yield of soluble hEGF. The suitable process conditions were tested for low cost production of hEGF in industry. Finally, we compared the results with those of previous studies to understand if this method can be industrialized. We reported high-level expression of soluble hEGF in recombination E. coli by HCDC, and the results have practical production value in this study.

Bacterial strain, equipment, and chemicals

Recombination E. coli LS-1 used in this study was constructed in our laboratory in Nanjing Tech University. The gene coding for the designed hegf was chemically synthesized by Sangon Biotech Co. Ltd. (Shanghai, China). The E. coli DH5α was used as the host strain for cloning vectors, and was the donor in translation as well. The E. coli BL21 (DE3) and plasmid pET-22b(+) were supplied by College of Food Science and Light Industry. Human HB-EGF ELISA Kit was purchased from Boster Biological Technology Co. Ltd. (Wuhan, China). Pre-stained Ultra Low Molecular Weight Protein Marker (3.3–31 kD) was obtained from Solarbio Co. Ltd. (Beijing, China). A controlled bioreactor (5 L) (BIOTECH-5BG-2) was provided by Baoxing Biological Equipment Co. Ltd. (Shanghai, China). A low temperature cooling liquid circulating pump was provided by Daweikejiao Equipment Co. Ltd. (Hangzhou, China). A biosensor (SBA-40C) was provided by Biology Institute Shangdong Academy of Sciences to enzymatically determine remnant glucose in the broth (Shandong, China). All other chemicals were pure reagents, and were available in our laboratory.

Medium

The inoculum was cultured in Luria-Bertani (LB) medium (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, ampicillin 100 µg/mL) or LB plate medium with 1.5% agar. The optimum flask medium was employed as a preliminary experiment to produce hEGF in shake flasks, containing glucose (5 g/L), yeast extract (20 g/L), K₂HPO₄ (14 g/L), KH₂PO₄ (6 g/L), NaCl (0.5 g/L), MgSO₄ (0.5 g/L), ampicillin (50 µg/mL), and 0.5 mM lactose to induce expression of the gene hegf. The fermentation medium contained glucose (5 g/L), yeast extract (10 g/L), tryptone (20 g/L), NaCl (0.5 g/L), and MgSO₄ (0.5 g/L) in a 5 L controlled bioreactor. All media, glycerol, lactose, and glucose were sterilized at 121 ℃ for 21 min, 108 ℃ for 30 min, 115 ℃ for 15 min, and 118 ℃ for 15 min, respectively.

Construction of the bacterial strain LS-1

The chemically synthesized gene hegf, carrying about 15 base pairs as homologous sequence with gene hegf on both ends, was amplified via PCR using in-fusion primers, pET-22b-F and pET-22b-R. The hegf and linear plasmid pET-22b(+) were ligated into plasmid pET-22b-hEGF using in-fusion cloning with restriction sites of NcoI and XhoI. Then, the recombination plasmid pET-22b-hEGF was transformed into competent E. coli DH5α strain. Finally, the plasmid pET-22b-hEGF was transformed into LS-1, which was formed by an expression host BL21 (DE3). The recombination LS-1 was preserved in 15% glycerol stocks at -80 ℃.

Preparation and cultivation of inoculum

A loopful of LS-1 glycerol stocks was inoculated into LB plate with 100 µg/mL ampicillin at 37 ℃ for approximately 18 h. Then, LS-1 colony was picked and cultured into a 20 mL shake-flask containing 5 mL LB media with 100 µg/mL ampicillin at 37 ℃ for approximately 6–8 h with shaking at 200 rpm. Primary seed inocula (3%) were added into a 250 mL shake-flask with 30 mL flask medium at 37 ℃ with shaking at 200 rpm, until the optical density was not less than 5 at 600 nm. Lastly, the secondary inocula were harvested from the 250 mL flask fermentation broth and used in a 5 L controlled bioreactor.

Preliminary fermentation in a shake flask

Preliminary fermentation experiments with a working volume of 30 mL were carried out in a 250 mL shake flask with shaking at 200 rpm. The fermentations of obtained soluble hEGF production were conducted at various induced temperatures and times in flask medium with an initial pH of 7.1 ± 0.1. A fermentation experiment was started by adding 3% primary seed inoculums at 37 ℃, fermented for 2.5 h, followed by induction of hEGF by addition of the ideal inducer (0.5 mM lactose) and simultaneously decreasing the temperature to 15, 20, 25, or 30 ℃. The optimal fermentation was terminated 17.5 ± 1 h after induction. Samples of 4 mL were centrifuged for 5 min at 4 ℃ at 8000 rpm. The rest of the broth was stored at 4 ℃ for using in further studies. The induction times (4, 8, 12, 16, 20, 24, 28, and 48 h) were investigated using the same method.

Strategy for pH-stat feedback feeding fermentation in a 5 L controlled bioreactor

To adjust the experiment scale, fermentation based on pH-stat feedback feeding (feeding medium: glucose 500 g/L) was carried out in a 5 L controlled bioreactor with 60% working volume (3 L) at either 37 ℃ or 32 ℃ at an initial pH of 7.1 ± 0.1. The agitation was controlled continuously at 700 rpm, and the ventilation was set between 1 and 3 vvm. The initial pH of fermentation medium was adjusted to 7.0 ± 0.1 using 2 M NaOH. In the experiments, the process pH of fermentation less than 7.0 was added using 2 M NaOH as well. The pH was maintained at more than 7.1 by addition of 500 g/L sterilized glucose during fermentation, which also ensured reasonable glucose concentration (The glucose concentration was kept in the range of 1–5 g/L for continuous growth of LS-1. However, acetic acid was formed, drastically decreasing the pH value, when the added glucose concentration was more than 5 g/L (Lecina et al. 2013; Eryasar-Orer & Karasu-Yalcin 2021).). Samples of 5 mL were taken at intervals of 2 h, and OD₆₀₀, glucose, and hEGF were determined using light absorption, SBA-40C, and ELISA kit, respectively.

Analytical methods

The optical density at 600 nm (OD₆₀₀) was used as a measure of bacterial strain LS-1 growth in the fermentation medium. Meanwhile, the fermentation broth was appropriately diluted from 10- to 200-fold with pure water before determination of OD₆₀₀. The relationship between OD₆₀₀ and dry cell weight (DCW) can be written using the following equation:

$$\text{y}=0.4066\text{x}-0.0006 {R}^{2}=0.999$$

where x is OD₆₀₀ value, y is DCW (g/L). Fermentation broth (4 mL) was centrifuged at 8000 rpm for 5 min at 4°C at the end of fermentation. The bacterial sludge fragmentation and inclusion body dissolution was performed according to a previously reported method (Clark 2001). The resulting samples were analyzed using modified sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Tricine-SDS-PAGE (Haider et al. 2018) was used to separate and qualitatively detect low molecular weight polypeptides or proteins (below 15 kD). In addition, the hEGF was quantitatively determined using an enzyme-linked immune sorbent assay (ELISA) with a diagnostic kit, which consisted of 96-well plate with pre-coated heparin-binding EGF (HB-EGF) antibody, biotinylated anti-human HB-EGF antibody, avidin-biotin peroxidase complex, and recombination human EGF standards. The hEGF samples were diluted from 10 to 100 million with a sample diluent. The formula of hEGF and OD₄₅₀ is given below:

$$\text{y}=0.0046\text{x}+0.0562 {R}^{2}=0.999$$

where x is hEGF concentration (pg/mL), y is OD₄₅₀ value.

Construction of strain E. coli LS-1

An efficiently engineered strain for biosynthesizing the human epidermal growth factor was constructed in this work. The plasmid pET-22b(+) was used as an expression vector to ligate gene hegf to form a new non-secretory plasmid pET-22b-hEGF in LS-1. Accuracy of the gene hegf, plasmid pET-22b(+), and pET-22b-hEGF was verified using PCR and agarose gel electrophoresis (Fig. 2a-c).

The supernatants obtained after lysis of recombinant strains were separated and qualitatively detected using Tricine-SDS-PAGE. A clear band with a molecular weight of about 7.0 kD of hEGF was visualized in lane one of Fig. 2d. Several reports showed that molecular weights of EGF were between 5.8 kDa and 7.5 kDa (Lee et al. 2006; Sonntag et al. 2014; Thomas & Walmsley 2014; Chen et al. 2017; Eissazadeh et al. 2017). The molecular weight of ~ 7.0 kD from the recombinant strain was consistent with the above-mentioned reports, because expressed polypeptides contained pelB signal peptides and six histidine-tagged peptides. In addition, we obtained hEGF at a yield of 45 mg/L using the supernatant from the engineered bacteria of LS-1 in LB medium. This production yield of soluble recombination peptides was much higher than that obtained in 0.5 L shake flasks by Norio Shimizu (Shimizu et al. 1991), indicating that LS-1 can be potentially used for commercial production of hEGF.

Effect of induced temperature on the expression of soluble hEGF in shake flasks

The effect of induced temperature on the expression of soluble hEGF in shake flask was investigated in this section. High temperature not only stimulates rapid growth of the recombinants, but also enhances the production of the target polypeptide hEGF. However, inclusion bodies could also increase greatly, complicating the purification process. Therefore, a choice-induced temperature is very importance for the expression of hEGF in E. coli. The results showed (Fig. 3a) that the maximum yields of soluble hEGF, inclusion bodies, and DCW were 76 ± 5 mg/L, 125 ± 20 mg/L, 7.02 g/L at 20 ℃ and 30 ℃, respectively. In general, low temperatures were not conducive for rapid growth of E. coli, but could be more benefit for expression of the exogenous polypeptides of low molecular weights (soluble) (Chen et al. 2012; Sajitha et al. 2015). Therefore, maximum DCW and inclusion bodies were obtained at 30 ℃. However, maximum expression of soluble hEGF was observed at 20 ℃. Moreover, DCW and total hEGF showed the lowest yields at 15 ℃. Many inclusion bodies were dissolved and determined using complex and expensive methods in the cytoplasm of strains. Fortunately, only at 20 ℃, a ratio of 54.3% of soluble hEGF and total hEGF was obtained. In contrast, a ratio of under 50% was computed at other temperatures. Therefore, establishing a valid technology for renaturation of inclusion bodies will be a key point in industrial production. Considering the complexity of the process of inclusion body refolding, a low temperature of 20 ℃ was selected for next investigation.

Effect of induced time on the expression of soluble hEGF in shake flasks

The induced time influences the expression levels of exogenous proteins or polypeptides during the fermentation process (Pinsach et al. 2008). Both inducer-added time and induced time were tested in this section. Figure 3b shows that soluble hEGF at a yield of 86 ± 5 mg/L was obtained adding inducer (L-lactose) to the shake flasks after fermentation 2.5 h, a total fermentation time of 20 h. Based on above results, various induction times of 4, 8, 12, 16, 20, 24, 28, and 48 h were carried out in the 250 mL shake flasks with 30 mL working volume. As shown in Fig. 3c, a gradual increase in the DCW and hEGF in supernatant was noted with an increase in induction times. A yield of 98 ± 10 mg/L in the supernatant was observed after 28 h of induction, and overall fermentation of 30.5 h, under the condition of the obtained maximum DCW (6.98 ± 0.3 g/L). The expression rate of polypeptides was higher with induction time of less than 16 h than with other induction times, mainly due to high activity of strains. Figure 3c also shows a slight increase in the number of hEGF on increasing the induction period from 16 h to 28 h, because the DCW also increased. Inducer-added time of 2.5 h and gene hegf-induced time of 28 h were selected as optimum for the next experiments.

Effect of factory-scale control on expression of soluble hEGF based on a pH-stat strategy in a 5 L bioreactor

It is difficulty to apply the identical process conditions from optimum parameters in flask shake to scale of plant-wide control. Low temperature (Wang et al. 2009) is used for induction of enzyme or low-molecular-weight polypeptides expression in the laboratory. As we know, even the temperature is decreased to 10–15 ℃ at a controlled condition in a lab-scale application (Baneyx 1999). Although the “Three high” are reduced via reasonable changed parameters in a pilot plant test, the cost of process is more in line with the benefits of the factory. This idea can be learned from a lab-scale a to pilot test for hEGF production.

Based on the above mentioned results and previous reports (Lindner et al. 2014), HCDC was carried out in a 5 L bioreactor with controlled pH-stat strategies to adjust for large scale production (Bordeaux et al. 2014). In this section, we mainly investigated the effect of induced temperature changing on the expression of soluble hEGF, and noted the optimum temperature that was consistent with plant-level control and showed maximum benefits. Therefore, 25 ℃ was chosen as an induction temperature suitable for large-scale control. As shown in Fig. 4a-b, adding 0.5 mM lactose in fermentation medium after 12 h was better than adding it after 2.5 h, at an induced temperature of 25 ℃. The yield of soluble hEGF was 328 ± 10 mg/L after 12 h of added inducer and 10 h of fermentation. This yield could basically meet the requirements of industrial production. However, during the process, culture temperature reduced to induced temperature, which was long term, untimely, and expensive on a large scale. Therefore, thermostatic culture and induction were designed to surmount these unfavorable factors. A selected temperature of 32 ℃ (Zheng et al. 2009) was investigated for feasibility of industrial production of hEGF. Figure 5 shows that soluble hEGF was obtained at a yield of 285 ± 10 mg/L after inducing temperature of 32 ℃ 8 h and overall fermentation of 20 h. In contrast to the results shown in Fig. 4b, overall fermentation time and induction time were shortened approximately by 2 h, respectively. The yield of soluble hEGF decreased to about 43 mg/L. However, this yield of soluble hEGF was still able to meet the production requirements. Based on reported by Tony et al (D'Amore et al. 1989), raising the fermentation temperature by 7 ℃ can save $42000. Considering the costs, therefore, a thermostatic culture at 32 ℃ for 8-h induction and 20-h overall cultivation was feasible for production of hEGF in a 5 L bioreactor.

Comparison of parameters for the feasible and economic production of hEGF

Hitherto, several studies have produced hEGF using the DNA recombination technology in host microorganisms like yeast and E. coli (Clare et al. 1991; Mei et al. 2017). We compared their host strains, fermentation parameters, and costs with those of this study, the results of which are shown in Table 1. The yield of group D hEGF was 870 mg/L, which was higher than those in other groups. However, the production time was not reported in that paper. Some studies suggested that the period of production of hEGF was extremely long using yeast as the host organism (Eissazadeh et al. 2017). In addition, basal and feeding medium in their experiments was very expensive. The economic benefits were reduced due to a long period of production resulting in expending a large number of feeding medium. Group A, B, and E show economic benefits, as compared with groups C and D, mainly due to the low cost of medium and high yield in actual operation. The yields of hEGF in groups A, B, and E were 240, 380, 285 mg/L, respectively. Although the productivity was high in group B, it required a dissolved oxygen (DO) level greater than 20%, involving higher expenses of a dissolved oxygen electrode for online monitoring. Additionally, the cost of energy consumption (30 ℃ shift to 38 ℃) was less than this study, but only costs 0.7 US dollars express per 1 g of hEGF, which was lower than group A-D. Briefly, this study not only avoid to control the DO and the temperature drop, but also keep the production cost low and enlarge the output, which can cater to industrial production request preferably.

Table 1

Parameters comparison, and the production of hEGF rough analysis of economy
Group	Microorganism	Plasmid	Culture parameter	DCW (g/L)	Yield (mg/L)	Productivity (mg/L/h)	Cost ($∙t^− 1)			Cost/ hEGF ($‧g^− 1)	Refer-ence
Group	Microorganism	Plasmid	Culture parameter	DCW (g/L)	Yield (mg/L)	Productivity (mg/L/h)	Basal medium	Feeding medium	Ind- ucer^b	Cost/ hEGF ($‧g^− 1)	Refer-ence
A	E. coli JM101	lacUV5par8egf	32 ℃, pH 6.0, DO > 20%	91	240	5.45	26.4	151.6	157	1.4	Zheng et al. 2009
B	E. coli BL21	pBLMVL2EGF	30 ℃, pH 7.0, DO > 20%, shift to 38°C for induction	26.6	380	13.82	90.9	763.3	─	2.3	Zhang et al. 2007
C	E. coli HB101	pTRLBTl	37 ℃, pH 7.0, DO controlled using 50% O₂	21	60	3	394.2	15016.2	~ 238	260.8	Shimizu et al. 1991
D	P. pastoris GS115	pPIC9K	30 ℃, pH 5.0	380^a	870	─	2674.6	1474.9	5.2	4.8	Lee et al. 2006
E	E. coli BL21	pET-22b	32 ℃, pH 7.0	57.4	285	14.3	89.7	110.6	0.7	0.7	This study
NOTE: Raw material costs are based on Chinese raw material prices calculated. Additionally, 1 $=6.78 RMB, other costs are considered equal.
^a 380 is the wet cell weight. ^b A ton of fermentation broth requires the amount of inducer, that is, the cost.

In this work, a non-secretory recombinant E. coli LS-1 was constructed in order to express hEGF. The two-stage process involved preliminary fermentations in shake flasks, which was later adjusted to large-scale fermentation in a 5 L controlled bioreactor based on pH-stat feedback strategy. A yield of 98 ± 10 mg/L of soluble hEGF and a cell density of 6.98 ± 0.3 g/L DCW in shake flasks were obtained. Furthermore, we obtained a yield of 285 ± 10 mg/L of soluble hEGF, a final cell density of 57.4 ± 2 g/L DCW, and hEGF productivity of 14.3 mg/L/h at 32 ℃ for 20 h in a 5 L bioreactor. Lastly, comparing the parameters for economically feasible production of hEGF, we conclude that we have developed an efficient and a low-cost production method of soluble hEGF.

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgments

This work was supported by the National Nature Science Foundation of China (21776133), the Six Talent Peaks Project in Jiangsu Province (SWYY-027), and the National Natural Science Foundation of China (Grant No. 21776075).

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Editorial decision: Major revisions
13 Aug, 2022
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02 Jun, 2022
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High-level expression of soluble human epidermal growth factor in Escherichia coli

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