Optimization of Extracellular Production of Recombinant Human Bone Morphogenetic Protein-7 (rhBMP-7) with Bacillus subtilis

Abstract

Extracellular production of recombinant human bone morphogenetic protein-7 (rhBMP-7) was carried out through the fermentation of Bacillus subtilis. Three significant fermentation conditions and medium components were selected and optimized to enhance the rhBMP-7 production by using the response surface methodology (RSM). The optimum values of the three variables for the maximum extracellular production of rhBMP-7 were found to be 2.93 g/l starch, 5.18 g/l lactose, and a fermentation time of 34.57 h. The statistical optimization model was validated with a few fermentations of B. subtilis in shake flasks under optimized and unoptimized conditions. A 3-L jar fermenter using the shake-flask optimized conditions resulted in a higher production (413 pg/ml of culture medium) of rhBMP-7 than in a shake flask (289.1 pg/ml), which could be attributed to the pH being controlled at 6.0 and constant agitation of 400 rpm with aeration of 1 vvm.

Introduction

Bone morphogenetic protein-7 (BMP-7; also called osteogenic protein-1), a member of the BMP subfamily of the transforming growth factor β (TGF-β) superfamily, is a low molecular weight glycoprotein that belongs to the group of bone matrix proteins [13]. It has a strong effect on bone induction and can increase the expression of alkaline phosphatase (ALP) in cells [10,22].

Recombinant human BMP-7 (rhBMP-7) has been produced from mammalian cell expression systems, such as Chinese hamster ovary (CHO) cells [22], or Escherichia coli using a pET expression system [2,19,26]. However, the production yield of the rhBMP-7 from the mammalian cell cultures has been low and expensive. E. coli systems were also used for the production of rhBMP-7, but the formation of inclusion bodies and improper protein-folding has occurred during the process of protein expression and purification [19]. The refolding and assembly procedure of the protein is complicated, so E. coli systems are not encouraged for the mass production of rhBMP-7.

An alternative expression host for the large-scale production of foreign proteins is the gram-positive bacterium Bacillus subtilis. Compared with E. coli, B. subtilis is an organism free of any endotoxin, and it offers an efficient secretion apparatus that guides the expressed protein directly into the culture media [12,20]. The secretion of proteins in a B. subtilis system is influenced by the genetic, cellular, and culture conditions [6,8]. For example, recombinant nattokinase was produced in culture broth by B. subtilis with a plasmid [7]. In our previous study, we constructed a shuttle vector containing a human bmp-7 gene and a constitutive promoter. [16]. The plasmid was transformed into Bacillus subtilis. B. subtilis with the plasmid produced the recombinant protein rhBMP-7 within cells and then secreted it into the culture medium. The production and secretion of rhBMP-7 are influenced by certain factors such as the fermentation conditions.

In this study, our work continued for the design and optimization of simple and cost-effective fermentation conditions for the extracellular production of rhBMP-7 in B. subtilis by using statistical techniques, such as two-level factorial design and response surface methodology (RSM) [15]. A two-level factorial design was used to investigate the effects of fermentation conditions and medium components on the extracellular production of rhBMP-7, and to select a few significant ingredients. Based on the selected fermentation conditions and medium components, an optimal statistical model was established by the full factorial central composite design (CCD) [1] and then validated through a few fermentation experiments with B. subtilis in shake flasks and a jar fermenter.

 

Materials and Methods

Bacterium and Plasmid

The bacterium employed in this work was Bacillus subtilis containing plasmid pBPT62. The multicopy plasmid pBPT62 was constructed using the plasmid vector pLip and the gene bmp-7 cDNA from hMU000489 (Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea: GeneBank BC008584) [16]. The rhbmp-7 gene in the plasmid was expressed constitutively under the control of the Hpa II promoter [3]. The transformation of B. subtilis with plasmid pBPT62 was then carried out according to the conventional method [11,25].

Fermentation of B. subtilis

The stock culture of B. subtilis was stored in 50% (v/v) glycerol at -70℃. Prior to each fermentation experiment, the recombinant cells were taken from the frozen stock culture and subcultured onto an agar slant containing LB medium (5 g/l yeast extract, 10 g/l tryptone, 10 g/l NaCl) with kanamycin (50 μg/ml).

For the preparation of the inoculum, a single colony from the agar slant was transferred to 5 ml of the LB medium containing kanamycin (50 μg/ml) in a test tube and incubated for 15 h at 30℃ and 200 rpm. After reaching an optical density (OD) of about 1.0 at 600 nm, 1 ml of the inoculum was used to inoculate the main culture in a shake flask at 30℃.

For the optimization of the extracellular production of rhBMP-7 with B. subtilis, a number of fermentation experiments were performed at 30℃ and 200 rpm in 500 ml shake flasks containing 100 ml of the production medium in a shaking incubator (JeioTech Co., Daejeon, Korea). The production medium consisted basically of the LB medium, due to energy requirement of the plasmid, and a few carbon sources such as starch and lactose.

A 3-L jar fermenter (KoBiotech Co., Incheon, Korea) with working volume of 1.20 L was also employed for the fermentation of B. subtilis, at 30℃ with aeration of 1 vvm at 400 rpm for 48 h. The cell growth was determined by measuring the OD at 600 nm by a UV/Vis spectrophotometer (Thermo Co., Finland). The dry cell mass concentration was determined offline by weight and correlated linearly to OD600 (i.e., 1.0 OD600 = 0.265 g/l dry cell mass).

Analysis of Extracellular rhBMP-7

The culture broths were centrifuged at 12,000 ×g for 10 min at 4℃ in a bench-top centrifuge, and the cell-free culture supernatants were employed to determine the quantity of extracellular rhBMP-7 produced during each set of fermentations. The concentration of extracellular rhBMP-7 was measured using ELISA kits (R&D Systems Co., Minneapolis, MN, USA) following the manufacturer’s instructions.

Experimental Design

A series of statistically designed studies were performed to investigate the effects of the fermentation conditions and medium components on the extracellular production of rhBMP-7. First, a two-level factorial design was employed to select the important components among the fermentation conditions and medium components. Based on the results obtained in our preliminary experiments, seven variables (i.e., the fermentation time, initial pH of the medium, and lactose, starch, yeast extract, tryptone, and NaCl concentrations) were found to be the major variables involved in the extracellular production of rhBMP-7. Each variable was studied at two different levels, high and low, and the center point, which is the midpoint of each factor range. The minimum and maximum ranges of the seven independent variables are listed in Table 1 with respect to their actual and coded values.

The seven variables (i.e., factors) were analyzed by a 2N-2 twolevel factorial design scenario available in the statistical software package Design-Expert (ver. 7.1.1; Stat-Ease, Inc., Minneapolis, MN, USA), as indicated in Table 2. The first-order model used to fit the results of the two-level factorial design is represented as

where Y is the predicted response (i.e., the extracellular concentration) (pg/ml) of the rhBMP-7; Xi is the coded level of the independent variable; k0 is the intercept; and ki is the linear coefficient. The most significant factors (variables) influencing the production of rhBMP-7 by B. subtilis were chosen to evaluate the maximum production of rhBMP-7 by the lowest P-values (probability values). The steepest ascent design was used to determine the direction toward the predicted higher responses.

A full factorial central composite design (CCD) is usually used to acquire data to fit an empirical second-order polynomial model. The second-order model equation used to simulate the experimental data was as follows:

where Y is the predicted response; Xi and Xj are the coded levels of the independent variable; b0 is the design factor of interest; and bi and bij are coefficients. A combination of factors, (Xi)(Xj), represent an interaction between the independent factors.

Table 1.Ranges of seven variables used in the two-level factorial design.

Table 2.aData are shown as the mean ± SD (n = 3).

A full second-order polynomial model obtained by a multiple regression technique for two factors, using the statistical software package Design-Expert 7.1.1, was adopted to describe the response surface. The response surface graphs indicate the effect of the variables individually and in combination, and determine their optimum levels for the maximum extracellular production of rhBMP-7 with B. subtilis.

 

Results

Growth of B. subtilis and the Production of rhBMP-7

Recombinant microorganisms are fermented in nutritionally rich media for the replication of heterologous plasmids and the expression of foreign genes [28]. Therefore, the culture medium components and process parameters, such as the pH and temperature of the medium, should be taken into account when optimizing the production of a foreign protein with recombinant microorganisms. In our preliminary fermentation studies with B. subtilis, we investigated the dynamics of cell growth and extracellular production of rhBMP-7 in various culture media. Fig. 1 shows the growth of B. subtilis and its extracellular production of rhBMP-7 in a shake flask with LB medium. B. subtilis grew exponentially for the first 18 h and then the growth rate remained almost stationary for up to 42 h. The maximum extracellular production of rhBMP-7 by the cells was observed at 21 h, and it decreased after 21 h. The maximum extracellular production of rhBMP-7 occurred after the exponential cell growth phase.

Fig. 1.Growth of B. subtilis and its extracellular production of rhBMP-7 in a shake flask with LB medium. Data are shown as the mean ± SD (n = 3).

For fast cellular growth and efficient rhBMP-7 production with B. subtilis, various attempts were also made to screen a few process parameters during its fermentation with LB medium by using the one-factor-at-a-time method. Some amount of the rhBMP-7 expressed within cells was effectively secreted into the culture medium [16]. Thus, the maximum secretion of rhBMP-7 in culture medium was influenced by cell growth (i.e., fermentation time). The culture medium pH has played an important role for cell growth with B. subtilis [27] and also influenced the gene expression from B. subtilis [9]. Lactose and starch were well known as the preferred carbon sources for B. subtilis [5,24]. Yeast extract and tryptone in LB medium were also selected as energy sources for gene expression in this study, including the inorganic salt NaCl. As a result, two fermentation conditions (fermentation time and initial medium pH) and five medium components (lactose, starch, yeast extract, tryptone, and NaCl) were investigated for their effects on cell growth and the extracellular production of rhBMP-7.

Selection of Significant Fermentation Conditions and Medium Components by Two-Level Factorial Design

A two-level factorial design was used to evaluate the significance of the seven factors for the extracellular production of rhBMP-7: the fermentation time, initial pH of the medium, and starch, lactose, yeast extract, tryptone, and NaCl concentrations. The results obtained for the production of rhBMP-7 by 27-2 factorial design experiments are shown in Table 2. A statistical analysis, ANOVA, of the responses was also performed and the results are presented in Table 3. The effect, sum of squares (SS), F-value, P-value, and confidence level (%) for each factor were evaluated on the basis of the observed Y values (the rhBMP-7) in Table 2. The factors were selected at a confidence level of 95 % on the basis of their effects. The confidence levels for the initial pH of the medium (X2), and yeast extract (X5), tryptone (X6) and NaCl (X7), concentrations in the production of rhBMP-7 were all below 95%, and were hence considered insignificant.

The addition of yeast extract, tryptone, and NaCl to the LB medium did not affect the production of rhBMP-7 significantly (with a large P-value). Furthermore, they had a little negative effect, implying that the rhBMP-7 production decreased with increasing their concentrations in the LB medium. The change in initial medium pH between 6 and 8 also had no impact on rhBMP-7 production. By contrast, the variables fermentation time (X1), and starch (X3) and lactose (X4) concentrations showed confidence levels of 99.99%, 99.99%, and 99.23%, respectively, and were considered significant.

Table 3.SS, sum of squares.

From the results of the ANOVA in Table 3, the four factors of initial pH of the medium (X2) and yeast extract (X5), and the tryptone (X6) and NaCl (X7) concentrations were fixed at pH 6.0, 5.0 g/l, 10.0 g/l, and 10.0 g/l, and omitted as variables in the regression analysis for a firstorder polynomial model. Therefore, the first-order polynomial equation for the extracellular production of rhBMP-7 (Y) was derived using three factors - the fermentation time (X1), and starch (X3) and lactose (X4) concentrations:

Using this model equation, the Y-values were predicted as shown in Table 2. Thereafter, the exact optimum values for the three selected factors were determined using a fullfactorial CCD.

Optimization of the Selected Variables Using Response Surface Methodology

The CCD with five coded levels shown in Table 4 was used to find the optimum values of the selected factors (X1, X3, X4) for the extracellular production of rhBMP-7. To study the combined effects of these three factors on the production of rhBMP-7, a CCD consisting of 20 (23 = 8 + 6(center points) + 6(star points)) experiments was performed, as shown in Table 5. By applying multiple regression analysis to the experimentally observed rhBMP-7 data shown in Table 5, the experimental results of the CCD were fitted with a second-order polynomial model for the extracellular production of rhBMP-7. The adequacy of the model equation was checked using ANOVA, which was tested with Fisher’s statistical analysis, as shown in Table 6.

Table 4.Coded levels of significant independent variables selected for CCD experiments.

The ANOVA of the second-order polynomial model shown in Table 6 is highly significant, as is evident from the Fisher’s F-test with a very low probability {(Prob(P) > F) = 0.0001}. The fitting value, termed the R2 value (multiple correlation coefficient) of the model equation, was calculated to be 0.976, indicating that 97.6% of the variability in the response could be explained by the second-order polynomial prediction equation given in Eq. (2). The “adjusted R2” and “predicted R2” values were 0.955 and 0.929, respectively, which also indicate that the model is good (for a good statistical model, the R2 value should be in the range of 0-1.0, and the nearer to 1.0 the value is, the fitter the model is deemed to be). The present model also had an “adequate precision value” of 23.137, and this suggests that it can be used to navigate the design space. The “adequate precision value” measures the signal-to-noise ratio (SNR), and an SNR greater than 4 is essential for a model to be a good fit. The model also had a very low value of the coefficient of variation (CV = 6.49%), indicating a very high degree of precision and good reliability of the experimental data. The coefficients of the second-order regression model for the extracellular production of rhBMP-7 were calculated using Design Expert, and the following equation was obtained:

where Y is the response, rhBMP-7 concentration (pg/ml); and X1, X3, and X4 are the coded values of the selected variables (viz., the fermentation time (h), starch concentration (g/l), and lactose concentration (g/l), respectively).

Table 5.aData are shown as the mean ± SD (n = 3).

Table 6.R2 = 0.976; Adj-R2 = 0.955; Pred-R2 = 0.929; CV = 6.49%; SS, sum of square; DF, degree of freedom; MS, mean square.

From the ANOVA results shown in Table 6, the regression coefficients can be validated. The probability values (viz., the P-values) denote the significance of the coefficients and are also important in understanding the pattern of mutual interactions between the test variables. That is, values of “Prob(P) > F” less than 0.05 indicate that the model terms are significant. In this case, the model terms X1 , X3, X1X3, X1X4, X12, X32, and X42 were significant, but X4 and X3X4 were not significant. The nonsignificant term, X4 and X3X4 can be eliminated from the model (Eq. (2)), because they had P-values greater than 0.1 and caused a decrease in the adjusted R2 value of the model.

The experimentally observed data for the extracellular production of rhBMP-7 were compared with the rhBMP-7 data predicted by model Eq. (2). The parity plot showed a satisfactory correlation between the observed and predicted values for the extracellular production of rhBMP-7.

In order to study the interactive effect of each selected variable and its optimum level for the extracellular production of rhBMP-7, the three-dimensional response surface graphs were constructed by plotting the response (the rhBMP-7) on the Z-axis against two of the independent variables, while maintaining the other variable at a fixed level. Fig. 2A shows the response for the interactive effect of the starch and lactose concentrations when the fermentation time was kept at 32.0 h. The maximum extracellular production of rhBMP-7 under these conditions was predicted to be 281 pg/ml, corresponding to a high amount of starch (3.0 g/l). This indicates the positive effect of a high concentration of starch on the extracellular production of rhBMP-7. The interaction between the lactose concentration and fermentation time when the starch concentration was kept at 3.0 g/l for the extracellular production of rhBMP-7 is depicted in Fig. 2B. An increase in the production of rhBMP-7 was observed with increasing fermentation time. From Figs. 2A and 2B the optimal lactose concentration was also observed to be between 5 and 10 g/l. In Fig. 2C, the response varied as a function of the starch concentration and fermentation time when the lactose concentration was kept at 5.0 g/l. The global maximum in rhBMP-7 production would be confined within the experimental range; that is, between 0.5 and 3.0 g/l in starch concentration and between 24 and 36 h in fermentation time.

The statistically optimum values of the selected variables were obtained by moving along the major and minor axes of the contour, and the response at the center point on the 3D plots and the optimum values of fermentation time (X1), starch concentration (X3), and lactose concentration (X4) were identified as 34.57 h, 2.93 g/l, and 5.18 g/l, respectively. Therefore, the optimum conditions for the extracellular production of rhBMP-7 with B. subtilis in this work were found to be 2.93 g/l starch, 5.18 g/l lactose, 5.0 g/l yeast extract, 10.0 g/l tryptone, 10.0 g/l NaCl, pH 6.0, and a fermentation time of 34.57 h. Under these optimized conditions of the variables for the fermentation of B. subtilis, the maximum extracellular production of rhBMP-7 was 282.3 pg/ml, which was predicted by the second-order polynomial equation, Eq. (2). This result showed a significant improvement in comparison with the highest response of 71.881 pg/ml obtained with LB medium, as shown in Fig. 1.

Fig. 2.Response surface curves of the extracellular production of rhBMP-7, showing the interactions between the (A) lactose and starch concentrations, (B) fermentation time and lactose concentration, and (C) starch concentration and fermentation time.

Batch Fermentation in Shake Flasks and a Jar Fermenter

Several fermentation experiments with B. subtilis were carried out in shake flasks and have validated the statistical model established in this work. Fig. 3 shows the time courses of the extracellular production of rhBMP-7 using optimized and non-optimized conditions. B. subtilis produced 289.1 pg rhBMP-7/ml at 35.0 h under the optimized fermentation conditions and culture medium components, (i.e., 2.93 g/l starch, 5.18 g/l lactose, 5.0 g/l yeast extract, 10.0 g/l tryptone, 10.0 g/l NaCl, and pH 6.0), while the model predicted the production of 282.3 pg rhBMP-7/ml at 34.57 h. When the concentrations of starch and lactose were changed to 2 and 6 g/l or 1.75 and 7.5 g/l, B. subtilis produced 268.8 and 250.2 pg rhBMP-7/ml at 35.0 h, respectively, corresponding closely to the amounts (i.e., 266.1 and 250.5 pg/ml) of rhBMP-7 predicted by the model. Further experiments were repeated, with the results having a standard deviation of less than 5%. This close agreement between the experimentally observed rhBMP-7 values and the statistically predicted rhBMP-7 values validated the present model.

Fig. 3.Time courses of the extracellular production of rhBMP-7 under the optimized and two other fermentation conditions (fermentation condition 1 contained 2 g/l starch and 6 g/l lactose, while fermentation condition 2 contained 1.75 g/l starch and 7.5 g/l lactose). Data are shown as mean ± SD (n = 3).

B. subtilis has also been fermented in a 3-L jar fermenter under the shake-flask optimized and non-optimized conditions; that is, Ferm0A (2.93 g/l starch, 5.18 g/l lactose, 5.0 g/l yeast extract, 10.0 g/l tryptone, 10.0 g/l NaCl, and pH controlled at 6.0) and Ferm0B (5 g/l lactose, 5.0 g/l yeast extract, 10.0 g/l tryptone, 10.0 g/l NaCl, and pH controlled at 6.0). The extracellular production of rhBMP-7 with B. subtilis is shown in Fig. 4. B. subtilis produced 413 pg rhBMP-7/ml at 34 h in Ferm0A, while it produced 350 pg rhBMP-7/ml at 33 h in Ferm0B. The production of rhBMP-7 in a jar fermenter was increased as compared with that in a shake flask (289.1 pg/ml). This increase may result from a few reasons. The jar fermenter was operated at constant pH (pH 6.0) throughout the fermentation. The pH control of the culture medium would increase the growth of B. subtilis and also cause the use of lactose for effective production of rhBMP-7 [16]. Aeration of 1 vvm and agitation of 400 rpm during the fermentation in the jar fermenter would supply sufficient oxygen to cells and enhance the contact between cell and culture medium. The enhanced contact might also increase the secretion of intracellular rhBMP-7 into the culture medium [14].

Fig. 4.Production of the extracellular rhBMP-7 during fermentation in a jar fermenter under different culture medium components: Ferm0A (2.93 g/l starch, 5.18 g/l lactose, 5.0 g/l yeast extract, 15.0 g/l tryptone, 10.0 g/l NaCl, and pH controlled at 6.0) and Ferm0B (5 g/l lactose, 5.0 g/l yeast extract, 15.0 g/l tryptone, 10.0 g/l NaCl, and pH controlled at 6.0). Data are shown as mean ± SD (n = 3).

 

Discussion

For the extracellular mass production of a recombinant protein in biotechnology, it is necessary to manipulate the microorganisms genetically, to optimize the culture medium and conditions, and to control the process. A Bacillus subtilis/Escherichia coli shuttle vector has been used to produce recombinant nattokinase in B. subtilis. The B. subtilis strain was also employed to design an optimum and cost-effective medium for the high-level production of recombinant nattokinase using the response surface methodology [21]. The amount of recombinant nattokinase in the supernatant of the culture process carried out in the optimized medium was about 5 times higher than that obtained in the non-optimized rich medium. Therefore, the optimization of the culture medium and fermentation conditions is one of the most important strategies for the cell growth and production of recombinant proteins in B. subtilis [23].

Prokaryotic cells (Escherichia coli) [19] and mammalian cell expression systems, such as CHO, BSC-1 (Monkey kidney epithelial cells) [22,26], or COS7 (Monkey kidney fibroblasts) [18] cell lines have been used to produce the recombinant protein rhBMP-7. The transfected cells yielded approximately 3- to 10-fold more of the mature BMP-7 [26]. The rhBMP-7 produced with recombinant E. coli using a pET expression system was strongly mitogenic for MCT3T3-E1 cells. Even though the productivity of rhBMP-7 with these prokaryotic or mammalian-based systems was high, the cell systems have some disadvantages, such as expensive culture medium, the formation of inclusion bodies, and complicated purification steps. Recently, human 293T cells with a bicistronic lentiviral vector have been used to produce recombinant human BMP-7 [4]. The secretion of rhBMP-7 to the culture medium occurred after 72 h of serum starvation, and its productivity was very high (4.5 μg/ml of cell culture). However, human cells require extensive efforts or higher costs for mass production.

The mature part of the human bmp-7 gene was cloned and expressed in B. subtilis in our previous study [16]. B. subtilis produced high amounts of the rhBMP-7 protein within cells, some of which have been secreted into the supernatants during fermentation. For the extracellular mass production of rhBMP-7, it is necessary to optimize the fermentation process with B. subtilis. In this work, three significant factors (i.e., the fermentation time and starch and lactose concentrations) among the culture medium components and fermentation conditions were selected for the extracellular production of rhBMP-7 using a two-level factorial design. The three selected variables were optimized using RSM experiments (i.e., CCD) to maximize the production of rhBMP-7.

From the ANOVA in Table 6, the model was predictive, and the predictive rhBMP-7 values were in close agreement with the experimental values. When using the culture medium components and fermentation conditions identified with the RSM analysis, the maximum extracellular production (289.1 pg/ml) of rhBMP-7 was increased by 4-fold in comparison with that obtained with LB medium (71.9 pg/ml) in a shake flask. The production of rhBMP-7 with B. subtilis in a jar fermenter (413 pg rhBMP-7/ml) was also higher than that in a shake flask (289.1 pg rhBMP-7/ml). This could be attributed to the better control of the process parameters [14].

The results shown in this study clearly open up the possibility of using B. subtilis for the extracellular mass production of rhBMP-7 in an effective and inexpensive way. Using the optimal culture conditions and medium, successful adaptation of an industrial process and further increase in the mass production of rhBMP-7 can also be attained in fed-batch fermentations with B. subtilis in parallel stirred bioreactors [17] or in a jar fermenter [8].