INFLUENCE OF INDUCTION CONDITIONS ON THE ...

2nd Mercosur Congress on Chemical Engineering 4th Mercosur Congress on Process Systems Engineering

INFLUENCE OF INDUCTION CONDITIONS ON THE OVEREXPRESSION OF CARBAZOLE DIOXYGENASE FROM Pseudomonas stutzeri IN RECOMBINANT Escherichia coli Ariane L. Larentis1,2*, Rebeca G. Costa2, Haryana Sampaio1,2, Orlando B. Martins2, Tito L. M. Alves1 1

Programa de Engenharia Química - COPPE - Universidade Federal do Rio de Janeiro 2 Instituto de Bioquímica Médica - Universidade Federal do Rio de Janeiro

Abstract. In this work, E. coli was used to overexpress the carbazole 1,9a-dioxyganase, the first enzyme of the carbazole degradation pathway in Pseudomonas sp. That enzyme depends on the coexistence of three components containing [2Fe-2S] clusters, CarAa, CarAc e CarAd, and the catalytic site for oxygen activation is a mononuclear iron domain present in CarAa. The importance of this enzyme is associated to the carbazole potential pollutant characteristics as a recalcitrant nitrogen heterocycle molecule, commonly found in petroleum and other fossil fuels, responsible for nitrogen oxide production in oil combustion. The genes corresponding to carbazole 1,9a-dioxyganase components from Pseudomonas stutzeri were cloned and expressed by salt induction in native form in E. coli BL21-SI, a host that allows the enhancement of the soluble fraction of overexpressed proteins, using the vector pDEST-14. The expression of these proteins was performed in different conditions of cell concentration, temperature and time induction, with the help of 2-level factorial experimental design. Expression of CarAa was enhanced by the induction at lower cell concentration and temperature and higher times. CarAc and CarAd presented inverse effects and the best expression condition tested was the standard one. The enzymatic activity was detected for the degradation of 20ppm of carbazole.

Keywords: Heterologous Expression, Salt Induction and Experimental Design.

1. Introduction The degradation of carbazole and their dibenzopyrrole derivatives, commonly found in fossil fuels and associated to nitrogen oxide production during oil combustion, has been widely investigated, in particular in the last decade, due to the strengthening of environmental policy required by Kyoto Protocol concerning the reduction of gases emissions. The presence of these aromatic nitrogenated compounds, of which carbazole is one of the major species, is characteristic of Brazilian oils. These species are deleterious to the refining process, because they are “poisons” to the catalysts and alter the quality of petroleum derivatives (Benedik et al., 1998). In spite of that, some carbazole-degrading bacteria, as Pseudomonas sp., are able to degrade carbazole. The first enzyme of the degradation route is carbazole 1,9a-dioxygenase (CarA), which depends on the coexistence of three components containing [2Fe-2S] clusters: CarAa, CarAc e CarAd. The catalytic site for oxygen activation is a mononuclear iron domain present in CarAa (Sato et al., 1997; Larentis et al., in press). Using these enzymes overexpressed in recombinant microorganisms is a strategy of current great biotechnological interest in the

*

To whom all correspondence should be addressed. Address: Programa de Química, COPPE, UFRJ - Centro de Tecnologia, Bl.G, 21945-970 Rio de Janeiro – Brazil E-mail: [email protected]

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2nd Mercosur Congress on Chemical Engineering 4th Mercosur Congress on Process Systems Engineering biorrefin field because of the enhancement of protein production in comparison to the wild system, which can also improve the degradation rates. The Gram-negative bacteria Escherichia coli is the most frequently used prokaryotic expression host for highlevel production of recombinant proteins, also in commercial scale. Among the many systems available for heterologous expression, it remains one of the most attractive due to its ability to grow rapidly and at high cell densities on inexpensive substrates, its well-characterized genetics and the availability of an increasingly large number of cloning vectors and mutant host strains (Bhandari and Gowrishankar, 1997; Baneyx, 1999). However, in spite of the extensive knowledge on the genetics of this system, there are some problems associated to the bacteria: inability to fold some recombinant proteins into their proper conformation (functional, active and soluble form), leading to insoluble inclusion bodies, heterologous protein degradation by host cell proteases, inability to perform many of the posttranslational modifications found in eukaryotic proteins, and the lack of an efficient secretion mechanism for releasing of recombinant proteins into the culture medium (Wall and Plückthun, 1995; Makrides, 1996; Weickert et al., 1996; Bhandari and Gowrishankar, 1997; Baneyx, 1999). Moreover, in E. coli the expression of complex proteins, with multiple subunits, disulfide bonds, or posttranslational modifications, is hampered by the absence of cell apparatus able to promote the modifications essential to ensure the protein biological functionality. Also, one of the characteristics of the overexpression of heterologous proteins in E. coli cytoplasm is the misfolding and the tendency to aggregation into insoluble structures, the so-called inclusion bodies (Wong et al., 1988; Baneyx, 1999). This happens because in high levels of heterologous expression the upper limit of the production of chaperones may be exceeded. Chaperones are proteins present in bacteria and yeasts that mediate the correct folding and secretion of expressed proteins and, as a consequence, they mediate the proteolytic action over others proteins (Kurland and Gallant, 1996). Though the formation of inclusion bodies is described as a disadvantage of the E. coli expression system, some authors suggest that the formation of those aggregates could simplify protein purification, although requiring another step of refolding in vitro, which can signify a drawback in the production of large quantities of biologically active proteins (Kim and Lee, 1996; Makrides, 1996; Weickert et al., 1996; Joly et al., 1998; Lilie et al., 1998; Baneyx, 1999; Swartz, 2001). Two main strategies of overexpression of a target recombinant gene in E. coli are commonly used (Bhandari and Gowrishankar, 1997): temperature induction of a phage λ promoter or induction of a lac promoter and its variants with IPTG (isopropyl-β-D-thiogalactopyranoside). On the other hand, many researches have been done to enhance the performance and versatility of the expression system of E. coli aiming to overcome the limitations presented by the bacteria. In order to better control the levels of heterologous expression in E. coli and to enhance its ability to express functional proteins in high levels, promoters with different characteristics have been developed, as well as different genetic strategies have been carried out. These strategies encompass the addition or co-expression of chaperones in high quantities (Kurland and Gallant, 1996; Baneyx, 1999), the use of fusion proteins (Makrides, 1996) and to favor protein secretion to the bacterial periplasm, where the environment is oxidizing and facilitates the proper folding of proteins and disulfide bond formation (Makrides, 1996; Weickert et al., 1996; Joly et al., 1998). 2

2nd Mercosur Congress on Chemical Engineering 4th Mercosur Congress on Process Systems Engineering Besides exploring the genetic characteristics of the different E. coli strains employed for the recombinant proteins overexpression, the optimization of process conditions and strategies are essential to enhance the yield in protein production (Waters and Neujahr, 1994). As the majority of heterologous proteins are intracellular in the recombinant bacteria, the productivity is proportional to final cell density and to specific productivity (Lee, 1996). Thus, one strategy commonly used to enhance the quantity of produced protein is growing the bacteria in high cell densities for reducing undesirable effects, as substrate inhibition, limitation of the oxygen transfer capacity, formation of metabolites that inhibit the growing (Waters and Neujahr, 1994; Korz et al., 1995; Seeger et al., 1995; Kwon et al., 1996; Lee, 1996; Guengerich et al., 1997; Wang and Lee, 1997; Joly et al., 1998; Schmidt et al., 1999; Madurawe et al., 2000; Gupta et al., 2001). In these systems, it can be obtained up to tenths of milligrams per liter of recombinant product concentration, depending on the expressed protein and on the process conditions employed. It is well documented in the literature that the feed strategy (Lee, 1996), aeration (Waters and Neujahr, 1994; Korz et al., 1995; Seeger et al., 1995; Lee, 1996; Bhattacharya and Dubey, 1997; Gombert and Kilikian, 1998; Alba and Calvo, 2000; Gupta et al., 2001), medium composition and carbon sources in the feed (Waters and Neujahr, 1994; Korz et al., 1995; Kwon et al., 1996; Lee, 1996; Wang and Lee, 1997; Madurawe et al., 2000; Gupta et al., 2001; Fuchs et al., 2002), temperature (Lee, 1996; Gupta et al., 2001), antibiotics concentration (Gupta et al., 2001) influence in the E. coli cultivations in high cell densities, as these factors allows controlling the cell metabolism and avoid undesirable product formation, besides controlling and/or avoiding the formation of inclusion bodies. Also, the cultivation in lower temperatures can reduce the formation of inclusion bodies, the product degradation and the responses to cell stress conditions (Waters e Neujahr, 1994; Seeger et al., 1995; Kwon et al., 1996; Madurawe et al., 2000). Based on the importance of the study of the influence of process variable on the expression of recombinant proteins, the aim of this work is to evaluate the effect of some induction conditions (time, temperature and cell concentration) on the expression of the components CarAa, CarAc and CarAd of the enzyme carbazole 1,9adioxygenase from P. stutzeri in E. coli BL21-SI. This strain presents NaCl-inducible protein expression under the control of T7 promoter and protease deficiency for minimizing heterologous protein degradation (Bhandari and Gowrishankar, 1997). The employment of a 2-level factorial experimental design allowed identifying the interaction between the investigated variables, which is not usually found in the studies of recombinant proteins expression.

2. Experimental The three components (CarAa, CarAc and CarAd) of carbazole 1,9a-dioxygenase from P. stutzeri ATCC31258 were expressed in E. coli BL21-SI with NaCl induction using the plasmid pDEST14 in two different constructions: one containing carAa gene and the other with carAc and carAd genes, besides ORF7 gene, whose encoded protein is not essential to dioxygenase activity. The cloning was described in details in Larentis et al. (2004).

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2nd Mercosur Congress on Chemical Engineering 4th Mercosur Congress on Process Systems Engineering 2.1. Expression E. coli BL21-SI harboring recombinant Expression Vectors pDEST14 with carAa and carAcAd genes were cultivated in 10mL LBON medium (1% bactotryptone and 0.5% yeast extract), in the absence of NaCl, with ampicillin 100µg/mL at 37°C for 16h (pre-inoculum preparation). The inoculum was prepared with 5% of preinoculum in 10mL de LBON with ampicillin (resulting in an initial absorbance of 0.2 at 600nm). The bacteria were grown for 1h30min-2h until reached the exponential phase (absorbance 0.5 or 0.8 at 600nm, depending on the experimental conditions), then NaCl was added until the final concentration of 0.3M was reached for proteins induction at 30 and 37°C and in 4 and 24h. 1mL samples (before salt induction and in the different experimental conditions) were harvested and the pellets were stored at -20°C. E. coli BL21-SI without any plasmid was used as negative control and also the not induced samples.

2.2. Preparation of Cell Extract The pellets from 1mL samples of expression were resuspended in the proper buffer and sonicated to obtain the cell extract. For SDS-PAGE analysis, the cell extract were prepared in GET (glucose 50mM, EDTA 10mM and Tris-HCl 25mM) and submitted to total protein concentration measurement by Bradford method (Bradford, 1976), with bovine serum albumin as standard. For activity measurements, the cell extract was done in Tris-HCl 50mM (pH 7.5).

2.3. SDS-PAGE 18% SDS-Polyacrilamide Gel Electrophoresis was performed with 20µg of cell extract in a Bio-Rad apparatus. Gel was stained with Coomassie brilliant blue R-250.

2.4. Protein Expression Analysis The areas of the bands in SDS-PAGE corresponding to each protein expression (42kDa CarAa, 12kDa CarAc and 36kDa CarAd) were quantified by the program QuantiScan 1.25 and calculated considering 100% for the area obtained in the standard condition employed for induction of recombinant proteins in E. coli (A600nm=0.5, T=37°C and t=4h). Statistical evaluation of the cell concentration, temperature and time effects over the induction of proteins were done by empirical modeling using the normalized variable at levels –1 (lower value of the experimental condition used) and +1 (high value of the experimental effect), by the program Statistica 5.0.

2.5. Measurement of enzymatic activity Carbazole dioxygenase activity of CarA expressed in the optimized conditions was detected by the degradation of 20ppm (120µM) of carbazole by gaseous chromatography in a Varian CP-3380 with FID detector and column CP-SIL5CB. NADH (500µM), FAD (1µM), 35µM of ammonium ferrous sulfate (Fe(II) source) and 50µM of ascorbic acid (reducing agent) were used in sonicated cell extract (300µL) of the recombinant strains harboring plasmids with genes carAa and carAcAd. Tween 20 was added to aid with the solubilization of carbazole in water. The degradation was verified after 15h of reaction at 30°C in 50mM Tris-HCl (pH 7.5). 4


3. Results and Discussion The proteins CarAa, CarAc and CarAd were expressed in E. coli BL21-SI by salt induction in different conditions of cell concentration (A), temperature (T) and time (t). Through SDS-PAGE analysis, it was possible to identify the strongest bands in comparison to not induced samples below 45 kDa band of LMW marker (CarAa and CarAd), and by the appearance of a band below 14kDa (CarAc and ORF7), compatible with expected sizes of each protein (Fig. 1). Expression results indicate the enrichment of cell extract in the desired proteins by induction of recombinant plasmids with salt.

ind 4h ind 24 h ind

un

ind 4h ind 24 h u n ind ind 4h ind 24 h ind

un

OD 0,8 37°C

BL21-SI

CarAcAd

CarAa

90kDa 60kDa 45kDa 30kDa 20kDa 14kDa

OD 0,5 37°C

ind

ind 4h ind 24 h ind un ind 4h ind 24 h ind

OD 0,5 30°C

un

h

ind

24

4h

un

ind

CarAa

OD 0,8 30°C

90kDa 60kDa 45kDa 30kDa 20kDa

14kDa

OD 0,8 30°C

un

ind 24

h

ind 4h

un

ind

CarAcAd

OD 0,5 30°C

ind 4h ind 24 h ind un ind 4h ind 24 h ind

OD 0,5 37°C


14kDa

Fig. 1. SDS-PAGE of BL21-SI harboring recombinant pDEST14 vectors with 20µg of total protein in each sample of cell extract of CarAa, CarAc and CarAd expression in different conditions of cell concentration, temperature and time. Strain BL21-SI without the cloning construction was used as the negative control. Proteins were stained with Coomassie brilliant blue R-250. The protein molecular weight standard is LMW (Amersham Bioscience).

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For the proteins expressed by salt induction, SDS-PAGE showed three bands of 42kDa, 36kDa and 12kDa, corresponding to the CarAa, CarAd and CarAc expected protein sizes, respectively (Fig. 1). Expression results showed the enrichment of the target protein in the cell extract. Expression levels obtained for site-specific recombination system and salt-inducible cells were similar to those obtained for well-known pUC cloning system with IPTG induction (Sato et al., 1997). The areas of the expression bands calculated for each induction condition are presented in Table 1 and were analyzed by statistical methods.

Table 1. Area of expression bands of proteins CarAa, CarAc and CarAd in the different experimental conditions of cell concentration, temperature and time induction with 0.3M of NaCl, calculated in relation to standard condition (A600nm=0.5, T=37°C and t= 4h).

Condition A600

T

t

CarAa

CarAc

CarAd

A=0.8 T=37°C t= 4h

1.0

1.0

-1.0

107.2

171.1

9.0

A=0.8 T=37°C t=24h

1.0

1.0

1.0

19.6

202.2

0.0

A=0.5 T=37°C t= 4h

-1.0

1.0

-1.0

100.0

100.0

100.0

A=0.5 T=37°C t=24h

-1.0

1.0

1.0

116.0

25.3

47.8

A=0.8 T=30°C t= 4h

1.0

-1.0

-1.0

112.2

56.1

151.8

A=0.8 T=30°C t=24h

1.0

-1.0

1.0

163.2

84.0

69.9

A=0.5 T=30°C t= 4h

-1.0

-1.0

-1.0

101.8

23.2

83.1

A=0.5 T=30°C t=24h

-1.0

-1.0

1.0

255.7

149.5

42.5

For the proteins CarAa and CarAd it was possible to identify the variables with greater influence from the results of experimental design and to propose an empirical model for expression of these proteins. For CarAc the analyzed variables were no statistically significant, once the values of standard deviation were higher than the values of the parameters, invalidating the proposed mathematical model. Models obtained for proteins expression (in terms of relative area to standard condition) are, respectively:

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2nd Mercosur Congress on Chemical Engineering 4th Mercosur Congress on Process Systems Engineering Expression of CarAa = 121.94 – 21.43 A600 – 36.26 T + 16.65 t + –25.81 A600.t – 34.55 T.t

(1)

Parameter standard deviation: 0.63

Expression of CarAc = 101.4 + 53.9 A600 + 46.5 T + 27.7 t + 70.1 A600.T + 1.8 A600.t – 49.4 T.t

(2)

Parameter standard deviation: 51.1

Expression of CarAd = 63.0 – 23.8 T – 23.0 t – 29.4 A600.T

(3)

Parameters standard deviation: 7.1

The analysis of the model presented in Eq. (1) shows that temperature and the combination of this variable with time induction present the higher influence over CarAa expression, followed by cell concentration and its synergism with time. T and A600 induction presented inverse effects, that is, higher levels of CarAa expression can be obtained for lower temperatures and cell concentration, while for greater induction times the expression levels were higher. CarAd expression presented higher influence of the interaction of cell concentration with temperature induction, followed by temperature and time, all of these variables with inverse effects on protein expression. The reduction of CarAd expression in all conditions with 24h of induction in comparison to 4h can indicate an effect of protein degradation. Differently from CarAa, for higher cell concentration on salt induction, the model indicates higher levels of CarAd expression, in a conjugated effect of A600 with the temperature. This behavior can de associated to a toxic effect of this heterologous protein to the cell. Although the model for CarAc expression was not statistically validated, one can realize that the behavior of the analyzed variables is the opposite of the effects for CarAd expression: higher cell concentration, temperature and time seems to enhance the expression of this protein. The only inverse effect is the interaction between temperature and time. Through analysis of the influence of cell concentration, temperature and time over expression of carbazole 1,9a-dioxygenase components and the obtained models, protein expression could be optimized. The model in Eq. (1) indicates the induction of CarAa at A600nm of about 0.35, temperature of 25°C for 24h could enhance expression of the protein in 400% in comparison to standard conditions. The experiment for CarAa expression optimization was performed, confirming the analysis proposed in Eq. (1), as indicated in Figure 2. For CarAd, in the same temperature condition of 25°C, but at A600nm near 1.0, Eq. (3) indicates an increasing of 300% of expression of this protein in 4h of induction. However, as carAc gene is present in the same plasmid as carAd, and the expression of CarAc presents an inverse behavior in comparison to CarAd, as indicated in Eq. (2), CarAc was not expressed in the optimized condition proposed for CarAd (data not shown). It was concluded that the best expression results for both proteins were obtained from the standard condition (A600nm=0.5, T=37°C and t= 4h). The degradation activity of CarA for 120µM of carbazole at 30°C was confirmed in 15h of reaction of

cell extract of both recombinant plasmids obtained in the respective optimized expression condition.

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ins o lu bl e

sol ub le

ce

ex tra

tot al

un ind u

CarAa

d

ct

Induction OD 0.35 / 25°C / 24h


14kDa

Fig. 2. SDS-PAGE of BL21-SI harboring recombinant pDEST14 vector with 20µg of total protein in each sample of CarAa expression in the optimized condition of cell concentration, temperature and time. Proteins were stained with Coomassie brilliant blue R-250. The protein molecular weight standard is LMW (Amersham Bioscience).

The cell extract obtained in this optimized condition was also separated into soluble and insoluble fractions. The results indicated that, even with salt induction, in this condition the largest part of the recombinant proteins are expressed in insoluble forms, probably in inclusion bodies. The strain BL21-SI is derived from the strain GJ1158, which employs NaCl as inducer for the over expression of proteins. In this system, developed by Bhandari and Gowrishankar (1997), the synthesis of polimerase RNA from bacteriophage T7 (T7 RNAP) is controlled by the promoter proU from E. coli, osmotically induced. The expression levels in these strains are described as similar to those induced with IPTG, and salt induction is associated to the increase of proportion of overexpressed proteins in soluble fraction, because of significant reduction of inclusion bodies formation.

4. Conclusions Expression results showed the enrichment of the target protein in the cell extract for site-specific recombination system and salt-inducible cells, similar to expression levels obtained for the well-known pUC cloning system. The analysis presented in this work could help to propose experiments that enhance expression of target proteins. It was observed that each of the three components of carbazole dioxygenase presented different responses for each induction variable analyzed: expression of CarAa was enhanced by the induction at lower cell concentration and temperature and higher times; CarAc and CarAd presented inverse effects and the best expression condition tested in this work was the standard one. The enzymatic activity was also detected for the degradation of 20ppm of carbazole. The 2-level factorial experimental design was a valuable tool on the analysis of induction variables of the carbazole dioxygenase components expression and on the determination of optimized conditions. There are few works in the molecular biology literature that employ the experimental design tools in the expression of recombinant proteins, and it is usual to evaluate each variable separately, which may lead to a misinterpretation of obtained data, do not allow analyzing variables interactions, and usually require a greater number of experiments to reach the same conclusions found by the factorial design. 8


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2nd Mercosur Congress on Chemical Engineering 4th Mercosur Congress on Process Systems Engineering Madurawe, R. D., Chase, T. E., Tsao, E. I., Bentley, W. E. (2000). A Recombinant Lipoprotein Antigen against Lyme Disease Expressed in E. coli: Fermentor Operating Strategies for Improved Yield. Biotechnol. Prog., 16, 571. Makrides, S. C. (1996). Strategies for Achieving High-Level Expression of Genes in Escherichia coli. Microbiol. Rev., 60, 512. Sato, S. I., Nam, J.-W., Kasuga, K., Nojiri, H., Yamane, H., Omori, T. (1997). Identification and Characterization of Genes Encoding Carbazole 1,9a-Dioxygenase in Pseudomonas sp. Strain CA10. J. Bacteriol., 179, 4850. Seeger, A., Schneppe, B., Mccarthy, J.E.G., Deckwer, W.-D., Rinas, U. (1995). Comparison of temperature – and isopropylβ-D-thiogalacto-pyranoside – induced synthesis of basic fibroblast growth factor in high-cell-density cultures of recombinant Escherichia coli. Enzyme Microb. Technol., 17, 947. Schmidt, M., Babu, K. B., Khanna, N., Marten, S., Rinas, U. (1999). Temperature-induced production of recombinant human insulin in high-cell density cultures of recombinant Escherichia coli. J. Biotechnol., 68, 71. Swartz, J. R. (2001). Advances in Escherichia coli production of therapeutic proteins. Curr. Opin. Biotechnol., 12, 195. Wall, J. G., Plückthun, A. (1995). Effects of overexpression folding modulators on the in vivo folding of heterologous proteins in Escherichia coli. Curr. Opin. Biotechnol., 6, 507. Wang, F., Lee, S. Y. (1997). Production of Poly(3-Hydroxybutyrate) by Fed-Batch Culture of Filamentation-Supressed Recombinant Escherichia coli. Appl. Environ. Microbiol., 63, 4765. Waters, S., Neujahr, H. Y. (1994). A Fermentor Culture for Production of Recombinant Phenol Hydroxylase. Protein Expression Purif., 5, 534. Weickert, M. J., Doherty, D. H., Best, E. A., Olins, P. O. (1996). Optimization of heterologous protein production in Escherichia coli. Curr. Opin. Biotechnol., 7, 494. Wong, E. Y., Seetharam, R., Kotts, C. E., Heeren, R. A., Klein, B. K., Braford, S. R., Mathis, K. J., Bishop, B. F., Siegel, N. R., Smith, C. E., Tacon, W. C. (1988). Expression of secreted insulin-like growth factor-I in Escherichia coli. Gene, 68, 193.

Acknowledgments CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and CTPetro/FINEPo

N 21.01.0278.00 supported this work. We thank to Laboratório de Biologia Molecular (LBM), Laboratório da Professora Eleonora Kurtenbach (LABEK) and also Núcleo de Estudos de Genoma Johanna Döbenreiner/UFRJ staff for technical assistance.

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