Bioprocess Biosyst Eng DOI 10.1007/s00449-006-0099-3
ORIGINAL PAPER
Effect of bioactive peptides isolated from yeastolate, lactalbumin and NZCase in the insect cell growth Ronaldo Zucatelli Mendonc¸a Æ Elizabeth Cristina de Oliveira Æ Carlos Augusto Pereira Æ Ivo Lebrun
Received: 12 September 2006 / Accepted: 3 November 2006 Springer-Verlag 2007
Abstract In this study, we have described the biological activity of various hydrolysates and its effect on cell growth, growth rate and doubling time. A potent cell culture enhancer factor was observed in the yeastolate hydrolysates, mainly in the protein fractions with low molecular weight. In this case, a growth enhancer of 60.66% was obtained. Despite a lower efficiency of crude lactalbumin hydrolysates (14%), when lactalbumin and yeastolate were added together to the culture, the cell yields were of 102%, showing a synergic effect. Nevertheless, sub fraction from LMW, of lactalbumin, obtained by Sephadex G-10 gel filtration chromatography showed a higher positive effect (23.3%) than low molecular weight fraction of lactalbumin without this chromatography step (11.3%). It is suggested that low molecular weight lactalbumin could have some inhibitory protein. On the other hand, NZCase low molecular weight showed a positive effect of 29.33%, while its sub fractions showed a negative effect of 5.5%. With these data we can suggest that these hydrolysates could be an important element to design new media, serum free, being helpful in protein recombinant production. Keywords Insect cells Sf-9 Bioactive peptides Yeastolate Lactalbumin NZCase
R. Z. Mendonc¸a (&) C. A. Pereira Laborato´rio de Imunologia Viral, Instituto Butantan, Av. Vital Brasil 1500, CEP 05503-900 Sa˜o Paulo, SP, Brazil e-mail:
[email protected] E. C. de Oliveira I. Lebrun Laborato´rio de Bioquı´mica e Biofı´sica, Instituto Butantan, Sa˜o Paulo, SP, Brazil
Introduction The production of recombinant proteins in mammalian or in insect cell lines has been of great importance in public health. To this, the production of products in large-scale is needed. The maintenance of most mammalian cell lines in culture requires the addition of serum to the culture medium. The elimination of serum from mammalian cell culture is desirable since serum is expensive, hard to eliminate in downstream process and could be a potential source of contaminants. Various serum free mediums have been developed since this medium is costly. In order to eliminate this, many strategies have been performed, including the use of a basic medium supplemented with some essential elements. Ikonomou et al. [1] have recently published a review describing the importance of protein hydrolysates to cell culture performance. Protein hydrolysates could be an alternative to the large-scale serum-free culture of animal cells. Hydrolysates are complex mixtures of oligopeptides, polypeptides and amino acids that are produced by enzymatic or chemical digestion of casein, albumin, plant or animal tissues, or yeast cells [1]. Some animal tissues digested as Primatone RL [2, 3] were being seen as a cost-effective supplement under serum or serum-free conditions [2–5]. Schlaeger et al. [3] have shown that Primatone RL prolonged the stationary phase in mouse hybridoma culture, by delaying the apoptotic death of cells. Ikonomou et al. [4] have described a similar effect in Sf-9 and High-Five insect cell lines, where the stationary phase can be prolonged for 3–4 days. In this case, 3–7 · 106 cells/ml was obtained. Plants can also be a font of protein hydrolysates [6–9]. Ikonomou et al. [4] have suggested that soy hydrolysates (Hy-Soy)
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insect cell culture medium supplemented with lactalbumin hydrolysate and yeastolate has allowed growth of several species of fungus (Entomophthorales pathogenic). The research reported that the addition of salts to the basic medium of sugars plus lactalbumin hydrolysate and yeastolate caused a significant increase in biomass production of the three fungal species. Chan
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Viable cells /ml x 10 (5)
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crude yeastolate
crude lactalbumin
crude NZCase
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could be used in serum-free formulations for insect cells instead of meat hydrolysates. Nguyen et al. [10] have developed a fed-batch method that increased the density of insect cells (Spodoptera frugiperda, Sf-9 cells) by improving the yield of a recombinant protein produced by a baculovirus expression system. The feeding consisted of glucose, glutamine, yeastolate and lipids solution and obtained 1.2 · 107 cells per ml being obtained 20 mg l–1 recombinant protein (rhNGF). Bedard et al. [11] have shown that the additions of yeastolate ultra filtrate and an amino acid mixture permitting the production at higher cell density, led to increases in the volumetric yield of a recombinant protein (beta-galactosidase). Together, or separately, the concentrates, when added to uninfected late exponential phase cultures, lead to a doubling of the maximum total cell protein level normally supported by un amended medium. Donaldson and Shuler [7], have shown that BTI-Tn5B1-4 insect cell line can grow in a medium based on IPL-41 basal medium, containing Hy-Soy protein hydrolysate, yeastolate ultra filtrate, a lipid-sterol emulsion and Pluronic F-68, with virtually identical growth rates obtained in a serumfree media such as ExCell 405 (6.0 · 106 cells/mL) supporting the production of secreted human alkaline phosphatase (SEAP) at a volumetric level of about 65– 70 mg/l. Thus, the less expensive ISYL medium can deliver acceptable performance and may be suitable for large-scale insect cell cultures. Chan et al. [12], tried to optimise a cell culture yield by manual addition of a multi-component nutrient feed to batch culture. This nutrient feed was based on yeastolate ultra filtrate, lipids, amino acids, vitamins, trace elements and glucose. The production of beta-galactosidase was optimised with this. The optimum volumetric yield of beta-galactosidase in fed-batch culture was 2.4-fold than of the best yields in batch culture. Sung et al. [13] have used several low-cost hydrolysates such as yeast hydrolysate (YH), soy hydrolysate, wheat gluten hydrolysate and rice hydrolysate as medium additives to enhance the human thrombopoietin (hTPO) recombinant protein production. When 5 g l–1 hTPO YH was added, 11.5 times more of protein was obtained and culture longevity extended by 2 days. Ikonomou et al. [14] have reported the development of a new serum-free medium using a variety of hydrolysates, where the yeastolate ultra filtrate was found to have the most important effect on cell growth. Furthermore, Primatone RL was found to remarkably prolong the stationary phase of Sf-9 cell cultures. The maximal densities obtained were of 5.4 and 6.1 · 106 cells/ml, respectively. The stationary phase was prolonged for 3–4 days. Leite et al. [15] have showed that Grace’s
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Yeast + lac
Fig. 1 Effect of hydrolysates proteins (yeastolate, NZCase or lactalbumin) (a) and (yeastolate + lactalbumin) (b) upon Sf-9 cell culture. To determine the potential effect in cell growth, cell culture was supplemented at the inoculation time with 2% (v/v) of an crude solution (200 g/l). Cell viability and cell concentration were determined daily. The cultures were performed in a spinner flask (60 ml) using Grace’s medium at 29C and 100 rpm. The results are the average of three independent experiments
Bioprocess Biosyst Eng
et al. [16] have demonstrated that a single addition of nutrient concentrates (amino acids, lipids, glucose and yeastolate ultra filtrate) increased the yield of betagalactosidase by 2.4-fold. In this paper, we tested three complex materials largely used in cell cultures (yeastolate, lactalbumin and NZCase). These materials were partially purified in different fractions (high molecular weight and low molecular weight) in order to obtain a combination of factors that allows a better growing of cells and its reliability in this media.
Materials and methods Cell line, culture media and supplements The insect Sf-9 cell line (the American Type Culture Collection, 1711) was used in all the experiments. Cultures were seeded at a cell concentration of 3 · 105 cells/ml and cells were grown in 100 ml spinner flasks with a working volume of 60 ml and agitated at 100 rpm with a suspended magnetic bar impeller. Grace’s medium (Sigma), supplemented with 10% fetal bovine serum (Cultilab, SP) was used in all cultures. In the supplemented experiments, cultures were fed once at the inoculums with 2% v/v of crude or fractions (5% v/v) of yeastolate, lactalbumin or NZCase. For both, batch and 1X feed experiments, samples were collected at least daily, for quantification of cell numbers to determine the effect of these proteins in the growth rate, maximal cellular yields and survival time of the culture. Analytical methods Cell number was determined daily by counting under a microscope light using a Neubaeur haemocytometer. Viability was determined by trypan blue exclusion by counting 200 cells from each sample in a hematocymeter.
Molecular ultra filtration Ten ml of a 10% solution (w/v) of each media (yeastolate, lactalbumin or NZCase) in 0.02 M, pH 7.0 of sodium phosphate, was filtrated with an Ultrafree-15 (Millipore) molecular weight cut-off membrane 30 kD (this procedure was repeated six times), to obtain high molecular weight (HMW) and low molecular weight (LMW) fractions from the different media. Each media was centrifuged throughout molecular size exclusion (Ultrafree-15, Millipore) cut-off membranes with 30 kDa pore size at 3,000 rpm at 4C in an Incibra´sSpin centrifuge. To measure the amount of filtered material in each fraction, optical density was read at 280 nm in a spectrophotometer and then used in the assays with Sf-9 cells. Sephadex G-10 gel filtration chromatography Two ml of a 10% solution of each high and low molecular weight protein (yeastolate, lactalbumin or NZCase) in 0.02 M, pH 7.0 of sodium phosphate was applied to a Sephadex G-10 column (4 · 65 cm), previously equilibrated with sodium phosphate buffer solution (0.02 M, pH 7.0) and eluted with the same buffer with 12 ml/h flow rate. Fractions of 3 ml were collected by a LKB-Pharmacia Redifrac fraction collector and each fraction was read in 280 nm in a Micronal Model B382 spectrophotometer. Fractions corresponding to peaks were kept together in 4–6 pools that were lyophilized and stored at –20C. Protein of each pool was determined by Lowry (1951) method and compared to an albumin standard calibration curve.
Results In this study, the fed cultures were performed by a single addition of a variety of hydrolysates at the
Table 1 Cell culture parameters obtained after hydrolysates addition
l Td (h–1) Xmax Tmax (days) Cell growth enhancer (%)
Control 1
Yeastolate
Lactalbumin
NZCase
Control 2
Yeast + lact
0.020 34.65 21.4 06 –
0.031 22.35 34.8 06 62.6
0.029 23.90 28.5 06 33.1
0.023 30.13 18.6 05 –13.1
0.020 34.65 19.6 06 –
0.022 31.50 43.3 06 120.9
l Maximum specific growth rate (h–1) X max Maximal cell density (105 cells ml–1) Td (h–1) = Population doubling time (h) Tmax = time to reach maximal cell number
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inoculums. These supplements include, yeastolate ultra filtrate, NZCase and lactalbumin. The fed cultures were studied in terms of three significant variables, namely the maximal cell density, growth rate and time of stationary phase.
The results from the batch and fed culture with crude protein hydrolysates (yeastolate, NZCase and lactalbumin) are presented in Fig. 1a, illustrating the cell growth pattern. It can be noted that the cells allowed a maximal cell number around 6 days followed
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Viable cells /ml x 10 (5)
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Control
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5
Control
6 7 Days
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9
10 11
Lactalbumin HMW
Lactalbumin LMW
Yeastolate LMW
25
Viable cells /ml x 10 (5)
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15
10
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0 1
2
3
4
5
6
7
8
9
10 11
Days Control
NZCase HMW
NZCase LMW
Fig. 2 Effect of hydrolysates protein fractions of high and low molecular size (yeastolate, NZCase or lactalbumin) upon Sf-9 cell culture. To determine the potential effect in cell growth, cell culture was supplemented at the inoculation time with 5% (v/v) of hydrolysates fractions obtained after a molecular ultra
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filtration with an Ultrafree-15 (Millipore) molecular weight cut-off membrane 30 kD. Cell viability and cell concentration were determined daily. The cultures were performed in a spinner flask (60 ml) using Grace’s medium at 29C and 100 rpm. The results are the average of three independent experiments
Bioprocess Biosyst Eng Table 2 Cell culture parameters obtained after hydrolysates fractions of high and low molecular size addition
l Td (h–1) Xmax Tmax Cell growth enhancer (%)
Control Yeastolate HMW
Yeastolate LMW
Lactalbumin HMW
Lactalbumin LMW
NZCase HMW
NZCase LMW
0.0185 37.46 15.0 07 –
0.022 31.50 24.1 07 60.6
0.0195 37.0 17.2 06 14.6
0.019 35.59 16.7 07 11.3
0.022 31.50 14.2 06 –5.5
0.0235 29.49 19.4 07 29.3
0.0175 39.60 19.3 08 28.3
l Maximum specific growth rate (h–1) Xmax Maximal cell density (105 cells ml–1) Td (h–1) = Population doubling time (h) Tmax = time to reach maximal cell number
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Viable cells/ml x 10(5)
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0 1
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Control
pool 1
pool 3
pool 4
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pool 2
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pool 3
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25 20 15 10 5 0 1
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Control
Pool 1
Pool 2
Pool 4
Pool 5
Pool 6
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9
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11
Pool 3
Fig. 3 Effect of hydrolysates protein fractions of yeastolate (a), lactalbumin (b) or NZCase (c) upon Sf-9 cell culture. To determine the potential effect in cell growth, cell culture was supplemented at the inoculation time with 5% (v/v) of hydrolysates sub-fractions obtained after chromatograph in gel
filtration column. Cell viability and cell concentration were determined daily. The cultures were performed in a spinner flask (60 ml) using Grace’s medium at 29C and 100 rpm. The results are the average of three independent experiments
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Bioprocess Biosyst Eng Table 3 Cell culture parameters obtained after hydrolysates sub-fractions of low molecular size addition Yeastolate
l Td (h–1) Xmax Tmax (days) Cell growth enhancer (%)
Lactalbumin
Control
Pool 1
Pool 2
Pool 3
Pool 4
Pool 1
Pool 2
Pool 3
0.020 34.56 21.4 06 –
0.027 25.66 28.4 06 32.7
0.023 30.13 19.5 06 –9.88
0.021 33.00 19.7 06 –9.80
0.019 36.47 18.8 06 –12.2
0.025 27.72 26.4 06 23.3
0.021 33.00 22.8 06 6.54
0.023 30.13 20.2 06 –5.6
l Maximum specific growth rate (h–1) Xmax Maximal cell density (105 cells ml–1) Td (h–1) = Population doubling time (h) Tmax = time to reach maximal cell number
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(pool 1) of sub-fractions of yeastolate and lactalbumin, showed the higher growth enhancer effect (32.7 and 23.3%, respectively) on cell growth, being the responsible in the hydrolysates by the effect observed. In contrast, no NZCase peak showed an enhancer effect. Nevertheless, as in the previous experiments was observed a positive effect we can conclude that in this case, the not observed effect could be related to the sub-fraction concentration used in the experiment. A negative effect in cell growth is very clear with any of the sub-fractions. In the Fig. 4 and in the Table 3 is showed the effect of hydrolysates proteins sub-fractions of low molecular weight (yeastolate and lactalbumin) in the duplication time (td h–1) of Sf-9 cell culture. Cultures were supplemented at the inoculation time with 5% v/v of hydrolysates sub fractions obtained after chromatograph in gel filtration column. As can be observed, all sub-fractions (except pool 4 of yeastolate) showed some positive effect in cell growth rate and duplication time. Even in this case, the best results were
Yeastolate
pool 3
pool 2
Pool 1
pool4
pool 3
pool 2
pool 1
40 35 30 25 20 15 10 5 0 Control
td (h -1)
by a decline. As can be observed, the hydrolysate’s addition was followed by a higher cell yields, increasing by 62% in culture with yeastolate addition and 33% after lactalbumin supplementation. A significant increase of cell number in culture supplemented with NZCase was not observed. Besides, a significant increase in rate of growth (l) was observed after yeastolate (0.031) and lactalbumin (0.029) addition, in contrast to control culture (0.020). So, the duplication time was, 22, 35; 23, 90; 34, 65 h, respectively (Table 1). On the other hand, when the culture was supplemented with yeastolate and lactalbumin, a yield of 4.3 · 106 cells/ml was obtained, which is 120.9% higher than control culture Fig. 1b. After fractionation of hydrolysates by ultra filtration in two groups of proteins; one with high molecular weight and the other with low molecular size, it was observed that the enhancing effect was localized in the low molecular weight group of proteins. As described earlier about the crude protein, the higher effect was observed in the proteins from yeastolate. In this case, the yeastolate protein with low molecular weight enhanced the cell growth by about 60.6% in relation to control. Proteins of low molecular weight, obtained from NZCase, also showed a good enhancer effect (29.3%) (Fig. 2, Table 2). On the other hand, the disagreement among the results obtained from crude or fraction obtained from NZCase can be related to any inhibitory cell growth protein present in the crude NZCase. Fractions of high and low molecular weight from three hydrolysates were applied in a second chromatograph step (Sephadex G-10 gel filtration chromatography). To determine the potential effect in cell growth of these sub-fractions, Sf-9 cell culture was supplemented at the inoculation time with 5% (v/v) of each one. The results are presented in Fig. 3a (yeastolate), b (lactalbumin), c (NZCase). As can be observed in Fig. 3a, b and Table 3, the first chromatography peak
Lactalbumin
Fig. 4 Effect of hydrolysates protein fractions (yeastolate and lactalbumin) in the duplication time (td h–1) of Sf-9 cell culture. Cultures were supplemented at the inoculation time with 5% v/v of hydrolysates fractions obtained after chromatograph in gel filtration column. The cultures were performed in a spinner flask (60 ml) using Grace’s medium at 29C and 100 rpm. The results are the average of three independent experiments
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obtained after culture supplementation with sub-fraction of yeastolate of low molecular weight (pool 1).
Discussion Recently, many authors have described the biological activity of various hydrolysates and its effect on cell growth and protein productivity [1, 13, 15]. There is a lot of evidence that the hydrolysates act as an enhancer cell growth or as a growth factor or as an antiapoptotic protein. So, these hydrolysates can be an important element to design new media serum free, being helpful in protein recombinant production. In this paper, we have showed the presence of a potent cell culture enhancer factor in the yeastolate hydrolysates, mainly in the protein fractions with low molecular weight. In this case, a growth enhancer of 60.6% was obtained. Despite a lower efficiency of lactalbumin hydrolysates (14%), when lactalbumin and yeastolate were added together to the culture, the cell yields were of 102%, showing a synergic effect. Nevertheless, sub-fraction of pool 1 from lactalbumin LMW (Table 3) showed a higher positive effect (23.3%) than fraction of pool 1 from lactalbumin LMW (11.3%, Table 2). It suggested that LMW lactalbumin could have some inhibitory protein. As shown in Fig. 3, this inhibitory protein can be present in pool 3 (–5.6%). When NZCase-LMW proteins were added to cell culture, the cell growth yields increased to about 29%. In contrast, when all sub-fractions of LMW proteins were tested, an inhibitory effect was observed. At this moment, we are studying the biochemical characteristics of these proteins we intend to determine the mechanism of action and correlate this protein with other known proteins. As we showed earlier [17], glucose, fructose and lactate are depleted in Sf-9 cell culture after 6 days, when culture stays around 2 · 106 cells/ml. Despite this, differences observed in this study can be due to specific medium additives and not by limitation of nutrients or dissolved oxygen, since in supplemented experiment, an enhancer cell growth until 120% in relation to control culture was observed. So, the cell growth enhancer can be related to the use of other nutrient in medium culture as carbon source, maybe glutamine, after glucose depletion, by action of additives added to the culture. To this moment, no analyses of insect cell metabolism as a whole have been reported; proposing metabolic pathways and analyzing the role of each nutrient in this complex system when other exogen elements are used as additives. At this moment, we are looking for it.
By these results, we can conclude that protein hydrolysates have a positive effect not only on maximal cellular yields but also on growth rate. In this study, the best results were obtained with crude or purified protein from yeastolate. With these data, we could suspect the presence of any inhibitor proteins in NZCase hydrolysates, since after a partial purification of the crude hydrolysates a positive effect was observed, but after a higher purification step this disappears. We think that it can be related more to the protein concentration added than a lack of activities.
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Bioprocess Biosyst Eng tures of Chinese hamster ovary cells. In Vitro Cell Dev Biol Anim 37(9):549–559 14. Ikonomou L, Bastin G, Schneider YJ, Agathos SN (2004) Design of an efficient medium for insect cell growth and recombinant protein production. J Biotechnol 108(3):279–292 15. Leite LG, Alves SB, Batista Filho A, Roberts DW (2002) Effect of salts, vitamins, sugars and nitrogen sources on the growth of three genera of Entomophthorales: Batkoa, Furia, and Neozygites. J Virol Methods 105(1):87–98
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16. Chan LC, Young PR, Bletchly C, Reid S (2002) Production of the baculovirus-expressed dengue virus glycoprotein NS1 can be improved dramatically with optimised regimes for fed-batch cultures and the addition of the insect moulting hormone, 20-Hydroxyecdysone. J Virol Methods 105(1):87– 98 17. Mendonc¸a RZ, Palomares LA, Ramirez OT (1999) An insight into insect cell metabolism through selective nutrient manipulation. J Biotechnol 72:61–75
