Biotechnology Department, IPEN-CNEN, Av. Prof. Lineu Prestes, 2242, Cidade Universitária, 05508-900, São Paulo, Brazil
1 To whom correspondence should be addressed. e-mail: pbartoli{at}ipen.br
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Abstract |
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Keywords: Escherichia coli periplasm/human growth hormone/PL promoter/recombinant DNA/signal peptide
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Introduction |
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The major form of hGH (22 kDa) is synthesized from the hGH-N gene in the non-glycosylated form (Garcia-Barros et al., 2000), so prokaryotic expression systems such as Escherichia coli have been the choice host cells for the production of this recombinant protein. High-level expression of hGH has been obtained by using procedures known to optimize both gene transcription and mRNA translation and several groups have reported the expression of the hormone or of a derived fusion protein in E.coli cytoplasm (Goeddel et al., 1979
; Ikehara et al., 1984
; Dalböge et al., 1987
; Mukhija et al., 1995
; Shin et al., 1998
; Patra et al., 2000
). However, the cytoplasmic production of a protein has certain disadvantages: high-level accumulation often leads to insoluble protein aggregates that can be difficult to solubilize; a refolding step is frequently required to obtain the native conformation and to form the correct disulfide bonds; the protein of interest usually contains an N-terminal methionine (Becker and Hsiung, 1986
) that may play a role in antibody formation in patients treated with the hormone (Glasbrenner, 1986
). Alternative expression systems have been based on the secretion of the protein into the E.coli periplasmic space; in these systems, the protein is obtained in its soluble and perfectly folded form and can be more easily purified to homogeneity. Secretion, which mimics the natural process of the somatotropic cells in the pituitary gland, was achieved by linking the signal peptide sequence to the hGH gene. All signal sequences, composed of 1530 amino acids, are characterized by several charged amino acids at the N-terminus, hydrophobic amino acids in the central region and an amino acid with a short side chain at the cleavage site. It has been demonstrated that the signal sequence functions to facilitate the translocation of secretory proteins across the inner membrane. A membrane-bound signal peptidase then cleaves off the signal sequence to release the mature protein into the periplasmic space located between the inner and outer membranes of E.coli (Chang et al., 1987
).
Table I summarizes the most noteworthy periplasmic expression yields reported in the literature for hGH, together with the corresponding references and the analytical method utilized for the hormone determination. Except for our group, hGH quantification was performed by either RIA or ELISA, which has quite limited accuracy, especially in the case of complex mixtures like culture broth and crude extracts (Jacobson et al., 1997; De Oliveira et al., 1999
). Immunoassays, moreover, cannot discriminate between undesired hGH-related forms (e.g. mass and charge isomers) and the fundamental form of the hormone. Such discrimination is, however, now possible when one uses the more recently developed HPLC or free zone capillary electrophoresis (FZCE) methodologies that can also be directly applied to E.coli fermentation broth for both qualitative and quantitative purposes (Strege and Lagu, 1995
; McNerney et al., 1996
; Dalmora et al., 1997
; Jacobson et al., 1997
; Jorgensen et al., 1998
; Karlsson et al., 1999
; Ueda et al., 2001
; Soares et al., 2002
; Ribela et al., 2003
).
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Materials and methods |
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A cassette expression vector was obtained by introduction of the restriction endonucleases sites NdeI upstream of the starting codon by polymerase chain reaction (PCR), using as template a previously constructed expression vector (our reference vector) in which the hGH gene is preceded by a modified natural hGH signal peptide.
The synthetic primers used for this PCR were: forward primer, including the NdeI site and 6 nt of the hGH signal peptide (5'-GAG GAA TTC CAT ATG GGC TCC-3'); reverse primer, including a C-terminal sequence of the hGH gene and the BamHI site, downstream of the stop codon (5'-ATC GGA TCC TTA TCA GAA GCC ACA GCT GCC CTC C-3').
For the introduction of three different signal sequences the following synthetic primers were used. (i) DsbAhGH sequence: forward primer, including the NdeI site, 57 nucleotides of the DsbA signal peptide (underlined) and part of the hGH gene (5'-GAG GAA TTC CAT ATG AAA AAG ATT TGG CTG GCG CTG GCT GGT TTA GTT TTA GCG TTT AGC GCA TCG GCG TTC CCA ACC ATT CCC TTA TCC-3'); reverse primer, the same used for cassette expression vector construction. (ii) STIIhGH sequence: forward primer, including the NdeI site, 69 nucleotides of the STII signal peptide (underlined) and part of the hGH gene (5'-GAA TTC CAT ATG AAA AAG AAT ATC GCA TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAT GCC TAT GCA TTC CCA ACC ATT CCC TTA TCC-3'); reverse primer, the same used for cassette expression vector construction. (iii) npr2K5LhGH sequence: this was obtained with two PCRs. Reaction 1: synthesis of the forward primer utilized for reaction 2. Forward primer (5'-GAG GAA TTC CAT ATG GGT TTA GGT TTA GGT AAG AAA AAG AAA TTG TTA CTT CTC CTA CTG TCT AGT GCT GTC GCC GCT TCC TTT ATG-3'); reverse primer (5'-GGA TAA GGG AAT GGT TGG GAA GGC CTG AAC ACC CGG CAG ACT GAT GGT TAA ACT CAT AAA GGA AGC GGC GAC AGC-3'). Reaction 2: synthesis of the complete sequence npr2K5LhGH. Forward primer (product of reaction 1), including the NdeI site, 102 nucleotides of the npr2K5L signal peptide (underlined) and part of the hGH gene (5'-GAG GAA TTC CAT ATG GGT TTA GGT TTA GGT AAG AAA AAG AAA TTG TTA CTT CTC CTA CTG TCT AGT GCT GTC GCC GCT TCC TTT ATG GCT GTC GCC GCT TCC TTT ATG AGT TTA ACC ATC AGT CTG CCG GGT GTT CAG GCC TTC CCA ACC ATT CCC TTA TCC-3'); reverse primer, the same used for cassette expression vector construction.
After digestion of the cassette expression vector and the DNA fragments obtained with each PCR with the endonucleases NdeI and BamHI, the fragments were subcloned into the NdeIBamHI sites of the cassette, the resulting vectors being named DsbAhGH, npr2K5LhGH and STIIhGH (Maniatis et al., 1989).
Bacterial strains and cultivation
Escherichia coli RRI strain, carrying the low copy-number plasmid pRK248cIts encoding for the PL promoter repressor cI, was used as a primary host for transformation and propagation of plasmids. For hGH expression, the host strains W3110, HB2151, RB791, TP2339 and BMH7118 were also used. The last two strains were kindly donated by Sanofi-Synthélabo Recherche (Toulouse, France). The cultivation was carried out in 500 ml shake flasks containing 100 ml of LuriaBertani (LB) medium, which consisted of yeast extract (5 g/l), tryptone (10 g/l) and sodium chloride (10 g/l), or a richer culture medium (2x-HKSII) which was a 2-fold concentrate of the HKSII bioreactor medium utilized by Jensen and Carlsen (Jensen and Carlsen, 1990
), consisting of yeast extract (10 g/l), tryptone (20 g/l), acid hydrolyzed casein (4 g/l), salts (Mg, K and Ca) and trace metals (Fe, Zn, Mn, Cu, Co, B, Mo and I). Both media were supplemented with 100 µg ampicillin/ml. Each flask was inoculated with a single colony of E.coli cells harboring one of the described hGH expression vectors taken from LB agar plates and incubated on a rotary shaker at 30°C. After growth to different optical density (OD) levels, the culture was either maintained at 30°C or heat-induced at different temperatures as described.
Osmotic shock
Periplasmic osmotic shock fluid was obtained by the method of Koshland and Botstein (Koshland and Botstein, 1980). Briefly, a volume of fermentation broth corresponding to 100 A600 was harvested by centrifugation at 3000 g for 5 min. All subsequent steps were carried out at 4°C in an ice bath. Pellets were resuspended in 1.0 ml of ice-cold 10 mM TrisHCl, pH 7.5, containing 20% (w/v) sucrose. Then, 33 µl of 0.5 M EDTA, pH 8.0, were added and incubation on ice continued for 10 min. The cells were then centrifuged and the pellet rapidly resuspended by vigorous agitation in 1.0 ml of a cold 1 mM TrisHCl, pH 7.5, solution. The suspension was then incubated for 10 min on ice and centrifuged again for 5 min. The supernatant was removed and saved as the periplasmic fraction, also called osmotic shock fluid.
Analytical reversed-phase high-performance liquid chromatography (RP-HPLC)
A Shimadzu Model SCL-10A HPLC apparatus coupled to a SPD-10AV UV detector (Shimadzu, MD, USA) was used, employing the Class VP software, also from Shimadzu. The column was a C4 Vydac 214TP54 (25 cmx4.6 mm i.d., pore diameter of 300 Å and particle diameter of 5 µm) with a guard column (Vydac 214FSK54) between the sample injector and the main column and a silica precolumn packed with LiChrosorb Si-60, 7.912.4 µm (Merck, Darmstadt, Germany) located between the pump and the injector. All Vydac columns were purchased from Grace Vydac (Hesperia, CA, USA). The mobile phase consisted of 71% TrisHCl buffer (50 mM, pH 7.5) and 29% n-propanol, as described by Dalmora et al. (Dalmora et al., 1997), with a flow-rate of 0.5 ml/min, detector wavelength at 220 nm, column temperature maintained at 45°C and a sample volume of 25200 µl.
SDSPAGE
Discontinuous SDSPAGE, based on 15% polyacrylamide gels, was carried out under non-reducing conditions as described (Laemmli, 1970). Coomassie Brilliant Blue G-250 (USB, Cleveland, OH) was used for staining and the molecular mass markers were from Amersham Pharmacia Biotech (Piscataway, NJ).
Protein determination
The total protein present in the osmotic shock fluids was determined by a Micro BCATM Protein Assay Reagent Kit (Pierce, Rockford, IL), based on a detergent-compatible bicinchoninic acid formulation for the colorimetric detection and quantification of total protein in dilute aqueous solutions.
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Results |
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The influence of the length of the activation period on the hGH expression level was investigated in Erlenmeyer flask culture as shown in Figure 1. Escherichia coli RRI cells harboring the reference vector were grown in LB medium at 30°C. At an OD of A600 = 1.5, the temperature was increased to 42°C. The cell density value for starting activation was defined by preliminary experiments, then confirmed by the results presented below. After activation periods of 0, 2, 4, 6, 7 and 8 h, samples were withdrawn and the osmotic shock fluid was analyzed by RP-HPLC.
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Stage of induction
In order to examine the effect of induction at different cell densities on hGH expression, the E.coli RRI strain transformed with the reference vector was incubated at 30°C in LB medium. When the cell density reached ODs of 0.3, 0.5, 1.0 or 1.5, the temperature was increased to 42°C. After a 6 h activation period, the levels of hGH expression at each induction stage were compared, as shown in Figure 2. The expression levels progressively increased from 0.4 (activating at A600 = 0.3) to 1.4 µg/ml/A600 (activating at A600 = 1.5), but decreased significantly above A600 = 2.0 (data not shown). In the case of activation at low cell density, besides obtaining a lower expression, a higher amount of altered forms could also be seen upon RP-HPLC analysis. The two growth curves as presented in Figure 3 illustrate the different behavior occurring when the richer medium 2x-HKSII is used. In the latter case, the best expression was obtained upon activaton at A600 = 3.0 (data not shown).
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In order to examine the effect of the temperature of activation on hGH expression, the reference vector in the E.coli RRI strain, was incubated in either LB or 2x-HKSII medium at 30°C. The activation in the latter medium was initiated at A600 = 3.0, utilizing different temperatures between 30 and 45°C (Figure 4).
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Comparison between different media and with established bioreactor fermentation conditions
hGH expression, using the reference strain and the optimized conditions, was analyzed in LB medium or in the same richer 2x-HKSII medium described by Jensen and Carlsen (Jensen and Carlsen, 1990) that we used for bioreactor studies (De Oliveira et al., 1999
).
The cell-specific yield of hGH was practically the same in both media. Thus, with LB we obtained inter-assay statistics of 1.63 ± 0.65 µg/ml/A600 [coefficient of variation (CV) = 40%; n = 14], while with 2x-HKSII we obtained 1.56 ± 0.34 µg/ml/A600 (CV = 22%; n = 8). The main difference was in the growth curve, the final cell density after 6 h activation being almost 2-fold higher in 2x-HKSII medium: A600 = 6.1 ± 0.85 versus A600 = 3.5 ± 0.66. It is interesting to note that, in previous studies carried out in a 20 l laboratory bioreactor under controlled fermentation conditions and glucose feed (De Oliveira et al., 1999), the same reference strain provided 2.43 ± 1.09 µg hGH/ml/A600 (CV = 45%; n = 6), i.e. an expression level
50% higher when compared with Erlenmeyer cultivation in the same medium. It was later observed that the addition of 6 g/l glucose to the rich 2x-HKSII medium in Erlenmeyer flask culture practically inhibited all hGH expression, which went down to 0.07 µg/ml/A600.
Effect of different signal peptides on hGH expression
To examine the influence of the signal peptide on hGH secretion into E.coli periplasm, we constructed four vectors using different signal peptides. Our reference vector, as mentioned, is based on a eukaryotic signal peptide, derived from the natural sequence of the hGH gene. The second signal peptide utilized was from DsbA, a 21 kDa periplasm protein of E.coli which, because of its origin and function, should provide an efficient periplasmic secretion (Martin et al., 1993). The third was the npr signal peptide, modified by inserting a 2K5L cluster into the N-terminal portion to enhance positive charge and hydrophobicity: this same modification caused increased secretion of 20K hGH in E.coli (Uchida et al., 1987). The fourth signal peptide, heat-stable enterotoxin II (STII), is also from an E.coli protein and has already provided a highly efficient periplasmic secretion of hGH (Chang et al., 1987
). The hGH expression obtained with the four different signal peptides, using RRI as the host cell, is shown in Table II. The results show that the most efficient signal peptidehGH gene combination was that based on DsbA, with an
80% higher expression level compared with the reference vector.
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Since E.coli strains differ in their ability to promote the expression of cloned genes (Kaytes et al., 1986), the most efficient vector,
PLDsbAhGH amplified in RRI, was also used to transform the following strains: HB2151, W3110, RB791, TP2339 and BMH7118, their growth before activation being carried out at 30°C.
Activation was carried out, as already established, at a cell density of 3.0 A600 in 2x-HKSII medium. The expression levels obtained in E.coli W3110 and RB791 were significantly higher than those obtained using the same vector in RRI and 2.5-fold higher compared with the reference vector in RRI (Table III). It is also interesting to observe that W3110 was not only one of the most productive strains, but also provided the highest hGH mass fraction.
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Discussion |
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It is important to emphasize that the present study was carried out in shake flask cultures, a widely used laboratory procedure that, unlike bioreactors, seldom has been standardized for this purpose. When expressing different recombinant proteins in E.coli under the control of a thermally inducible promoter, numerous authors have recommended starting the activation (i.e. induction stage) at an OD between 0.2 and 1.2, without a description of the optimization procedure leading to this choice. The same considerations also apply to the reported range of the temperature of growth (from 28 to 32°C) and activation (from 36 to 45°C) and to the duration of the activation period of 14 h (Bernard and Helinski, 1979; Crowl et al., 1985
; Caulcott and Rhodes, 1986
; Crowl, 1986
; Breitling et al., 1990
; Lowman and Bina, 1990
; Cheng and Patterson, 1992
; Balakrishnan et al., 1994
; Love et al., 1996
; Heim et al., 1998
; Hoffmann et al., 1999
; Yin et al., 2003
).
In our study, the optimized period of activation found for shake flask cultures was 6 h, a result that is consistent with the observations of Simons et al. (Simons et al., 1984), Lowman and Bina (Lowman and Bina, 1990
) and Choi and Lee (Choi and Lee, 1997
) in their optimization of large-scale production of bovine growth hormone (bGH). The same optimum activation time was obtained by us in previous work employing the same reference vector for hGH expression, in which feed batch fermentation conditions were set up in a 20 l laboratory bioreactor (Dalmora et al., 1997
). Although it may be possible to greatly increase biomass by up to 1020-fold in a bioreactor under more controlled cultivation conditions (Makrides, 1996
), an increase of this order does not occur for the protein of interest. In our bioreactor, the cell-specific yield of hGH was only
50% higher than in shake flasks, with approximately the same inter-assay reproducibility.
Under our conditions, and in contrast to literature data, the optimized induction stage corresponded to A600 = 1.5 in the case of LB medium and to A600 = 3.0 for 2x-HKSII medium. In the case of LB medium, when activation was started below A600 = 1.0, a much lower yield of hGH (partly altered) was obtained, while above A600 = 2.0, the secretion yield also rapidly decreased. A similar situation was observed for the richer medium, though at higher A600 values. Figure 3 shows that the optimized stage of induction falls in the early log phase for both curves, as several authors recommend (Choi and Lee, 1997). This, however, corresponds to quite different OD values for the two media and obviously should be considered in any optimization process. Preliminary studies were also carried out with the use of our reference strain devoid of the plasmid encoding for the CI repressor but, at least up to 50 generations, no significant decrease in hGH expression was observed.
Concerning the temperature of activation, our data are in good agreement with the generally recommended literature value of 42°C. Above this temperature, hGH secretion yields decrease dramatically. Thus, to guarantee an acceptable inter-assay reproducibility, we preferred to set this parameter at 41°C to avoid losses as a result of occasional uncontrollable small temperature fluctuations. In the case of periplasmic prolactin (a much more labile protein hormone than hGH) for example, the activation temperature cannot exceed 38°C if good recoveries are to be obtained (data not shown). Quite interesting in this respect is the approach of several authors who describe activation at 4245°C for a short time (e.g. 520 min) followed by incubation for a longer period at 3741°C (Bernard and Helinski, 1979; Caulcott and Rhodes, 1986
; Balakrishnan et al., 1994
). There have been several reports, however, suggesting that induction temperatures below 42°C may be more suitable for protein production in general (Lowman and Bina, 1990
).
A medium richer than LB apparently had no influence on the cell-specific hGH secretion yield, but it did affect the hormone production per unit volume, since 2x-HKSII produced almost twice as much biomass as LB medium.
The effects of signal peptide changes on the secretion of bGH from E.coli were extensively studied by Klein et al. (1992) who failed to find any significant influence for the six alternative bacterial secretion signals investigated. In our case, on the contrary, one of the factors exerting the greatest influence on hGH secretion was the signal peptide. Simply switching from the natural hGH sequence to modified npr or to DsbA signal peptide provided statistically significant increases of 41 or 79%, respectively. The good performance of DsbA signal peptide for directing an efficient secretion and correct folding of a protein in bacterial periplasm has already been observed (Rietsch et al., 1996
; Hannig and Makrides, 1998
; Winter et al., 2000
). Quite unexpected was the poor performance we obtained with the STII signal peptide which, together with the phoA promoter, is responsible for the highest periplasmic expression ever reported for hGH (Chang et al., 1987
).
A statistically significant increase of hGH production (40%) was also obtained by switching from our RRI reference strain to RB791 or W3110, but keeping all other parameters unchanged, including the temperatures of growth and activation. The latter strain not only provided the highest hGH secretion in our study, but also the highest mass fraction, reaching 20% of the total periplasmic proteins, which can greatly facilitate downstream processing of the protein of interest. Escherichia coli W3110 has, in fact, already been referenced as one of the most efficient strains for hGH expression and periplasmic secretion (Chang et al., 1987
; Uchida et al., 1997
). No report could be found on hGH production in RB791, even though this strain has proven to be able to overproduce and excrete ß-lactamase or epidermal growth factor through the periplasm (Chalmers et al., 1990
).
In conclusion, we attained a statistically significant 150% increase in hGH periplasmic expression/secretion (i.e. a cell-specific yield of 3.9 µg/ml/A600) in an E.coli system based on the PL promoter after optimization of the period, stage and temperature of activation and the type of cultivation medium for four different signal peptides and six bacterial strains. Following the example of Yin et al. (2003
), who evaluated three different promoters and three host strains for the bacterial expression of a collagen-like polymeric biomaterial in shake flask cultures, and Choi and Lee (Choi and Lee, 1997
), who studied temperature, induction stage, expression time, IPTG concentration and host type, for bGH, we believe it is highly recommended that analogous optimizations always be carried out each time that a heterologous protein is expressed in a given host cell for use in scale-up production.
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Acknowledgements |
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Received June 20, 2003; revised September 17, 2003; accepted October 3, 2003