1 Institute of Zoology, Academia Sinica, Nankang, Taipei 11529 and 2 Department of Physics, National Dong Hwa University, Hualien 973, Taiwan
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Abstract |
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Keywords: chaperonin/fish/growth hormone/recombinant/refolding
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Introduction |
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Generally, at the global minimum energy level, the native structure of a protein is more stable, independent of its folding pathways (Anfinsen et al., 1961). However, the folding of a protein may follow specific pathways in vivo (Levinthal, 1968
). Secondary structures of a protein, such as
-helix, are fundamental motifs serving as the core for folding (Qian and Chan, 1996
). The secondary structures, unlike the tertiary interactions, reflect distinct, local physical characteristics of a protein (Dill and Chan, 1997
). It has been recognized in the zipper model (Schellman, 1958
) that partial cooperative interactions between a residue and its flanking residues play an important role in the transformation of coil residues to a helical structure (Hong and Schellman, 1992
). The small value of the initiation factor
also suggests that the initial step of protein folding is the rate-limiting step (Scholtz et al., 1991
). With this information, a rational experimental design such as a stepwise folding approach was made for folding processes (Chen et al., 1992
). Moreover, during stepwise folding, the intermediates are proposed to be in a `hierarchical' path (Baldwin and Rose, 1999a
,b
) with their conformation as a molten globule state, that could be detected via its optical properties (Ptitsyn, 1991
).
Growth hormone is a member of the protein family that also includes prolactin, somatolactin and placental lactogen. The functions of fish growth hormones are similar to those of mammalian growth hormones, both in vivo and in vitro. These include stimulation of the expression of rainbow trout IGF-1 (Moriyama, 1995), a downstream gene on the growth hormone stimulation signal transduction pathway (Hammerman 1989
) and stimulation of chum salmon peripheral blood leukocyte proliferation in vitro (Sakai et al., 1996
).
An X-ray crystallography study indicated that human growth hormone is a four-helix bundle protein that interacts with its receptors on cell surfaces via conformational interactions and hydrogen bonds (de Vos et al., 1992). The helical conformation of growth hormone offers a good model for monitoring their conformational changes during refolding processes.
In this paper, we demonstrate the refolding of two inactive recombinant fish growth hormones into functional forms, using a steric blocker and chemical chaperonin through a stepwise approach (Chen et al., 1992).
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Materials and methods |
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The secondary structures of recombinant growth hormones from the fish ayu (rPaGH) and yellow grouper (rEaGH), were determined using the PHDsec prediction program (Rost and Sander, 1993, 1994
; Rost, 1996
). For hydropathy analysis, both sequences were submitted to the bioinformatics server of the Weizmann Institute of Science, Israel, using the KyteDoolittle index values with window 17 (Kyte and Doolittle, 1982
).
Expression and induction of recombinant fish growth hormones
Recombinant E.coli harboring growth hormone gene of ayu (accession number: AF232710) or yellow grouper (accession number: AF232711) were grown in LB medium up to an OD600 of 0.3. Cells were then induced with 0.1 mM IPTG at 37°C for 16 h.
Purification of inclusion body
The inclusion body was isolated [modified from Giza and Huang (Giza and Huang, 1989)] from E.coli as follows. The induced cells were harvested by centrifugation at 4000 g for 30 min. The pellet was then suspended and mixed well with ice-cold lysis buffer 1 (50 mM TrisHCl, pH 7.8, 200 mM NaCl, 0.1 mM EDTA and 5% glycerol) followed by adding a 1/10 volume of lysis buffer 2 (50 mM EDTA and 10% Triton X-100). Additional lysozyme (final concentration 10 µg/ml) was added to facilitate cell wall breakage. The resulting mixture was put on ice for 3 h. The cell walls of E.coli were further collapsed by sonication. Cellular DNA was digested with DNase I (final concentration 0.3 µg/ml) at 37°C for 1 h. Total cell lysate was separated into soluble and insoluble portions by centrifugation at 4000 g for 30 min. The insoluble pellet was washed twice with ice-cold Milli-Q water. The entire lysis process was repeated to ensure complete lysis of the host cells.
Denaturation and renaturation of recombinant fish growth hormone
The pellet of precipitated protein was dissolved in denaturing buffer [4.5 M urea with 10 mM Tris base and 0.1 M ß-mercaptoethanol (ß-ME)] and the mixture was adjusted to pH 12 with NaOH, to dissolve the proteins completely. The precipitated substance was removed by centrifugation at 4000 g for 30 min. In the first refolding stage, the soluble protein was dialyzed against buffer 1 [10 mM Tris base, pH 11, 1 M urea, 0.1 mM ß-ME, 1 mM cysteine (Cys) and 5% glycerol] for 72 h at 4°C. The urea was then removed by dialysis against buffer 2 (10 mM Tris base, pH 11, 0.1 mM ß-ME, 1 mM Cys and 5% glycerol) for 24 h at 4°C. To neutralize the pH of the second refolding stage, the refolding mixture was dialyzed against buffer 3 (10 mM TrisHCl, pH 8.8, 0.1 mM ß-ME and 5% glycerol) for 4 h at 4°C. Finally, glycerol in the third refolding stage was removed by dialysis against buffer 4 (10 mM TrisHCl, pH 8.8 and 0.1 mM ß-ME) for 4 h at 4°C.
Quantitative analysis of the refolded protein
The extinction coefficient of bovine growth hormone (bGH) at 277 nm is 700 g-1 cm-1 ml (Burger et al., 1966). The biochemical characteristics of rPaGH and rEaGH are similar to those of bGH and hence the same coefficient was adopted for the estimation of protein concentrations.
Analysis of protein secondary conformation
CD spectra in the UV region were determined on a Jasco J-720C spectrophotometer at room temperature using a cuvette with a 0.1 cm light pathlength. The same solvent conditions were used for each refolded state CD scanning. Blanks (buffer without proteins) were used to eliminate the background absorption of buffers. Data are expressed as the molar ellipticity [] (° cm2/dmol), calculating the molecular weight of rPaGH as 22 607 Da and rEaGH as 22 274 Da.
Cell proliferation assay
The effect of refolded recombinant growth hormones on the stimulation of cell proliferation was examined by MTSformazan conversion (Promega). Zebrafish liver (ZFL) cells (Ghosh et al., 1994) were cultured in L15 medium (Life Technologies), supplemented with 0.5% fetal bovine serum and 0.1% bovine serum albumin, in 96-well plates and incubated at 28°C. Recombinant PaGH and rEaGH were added to the culture medium with final concentrations of 0 pM (control), 0.1 pM, 1 pM, 10 pM, 0.1 nM, 1 nM, 10 nM and 1 µM, to a final volume of 100 µl in each well. After incubation for 48 h, 20 µl of CellTiter 96 Aqueous One Solution (Promega) were added to each well and the plates were incubated at 37°C for 4 h in a humidified 5% CO2 incubator. The absorbance at 450 nm was measured by using a 96-well plate reader (CERES UV900 HDI, Bio-TEK Instruments). The relative proliferation ratio (%) of the rPaGH and rEaGH treated cells was calculated with respect to the absorbance at 450 nm of the control.
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Results |
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The structure and hydropathy analyses of amino acid sequences of rPaGH and rEaGH help in understanding the native physical biochemistry properties of fish growth hormones for the design refolding process. The predictions showed that both recombinant fish growth hormones, as mammalian growth hormone, have a four-helix structure. The predicted -helix region (PAR) of rPaGH from helix I to helix IV consists of residues from N4 to L33, V69 to L94, R98 to T120 and D148 to H193, respectively (Figure 1A
). Residues from G5 to S33, V70 to G94, R98 to Q120 and D147 to H191 are the four PAR of rEaGH, respectively (Figure 1B
). The helical structures facilitate the monitoring of the secondary conformation of intermediates during the folding process by using a CD spectrophotometer. Hydropathy analyses indicated that both recombinant fish growth hormones possess a hydrophilic propensity (Figure 1
). This information suggests that both recombinant proteins can be dissolved in low ionic strength solution.
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Both rPaGH and rEaGH were expressed in E.coli. The cell lysates were separated into soluble and insoluble fractions by centrifugation. Analysis of these fractions by SDSPAGE revealed that the majority of both expressed recombinant GH were in the insoluble portion (data not shown). The weights of inclusion bodies of rPaGH and rEaGH were about 70 and 30 mg/g E.coli, respectively. The weights of final folded rPaGH and rEaGH were 67 and 28 mg/g E.coli, with percentage yields of 96 and
93%, respectively. The molecular weights of rPaGH and rEaGH by SDSPAGE were
28 and
22 kDa, respectively.
Denaturation and refolding
Both rPaGH and rEaGH expressed from E.coli are insoluble until in the high pH denaturing solution. Upon complete dissolution, the recombinant fish growth hormones were subjected to renaturation by a four-step equilibrium dialysis procedure. The denaturing reagent urea, high pH value and chemical chaperonin glycerol were removed stepwise.
Optical analysis
A CD spectrophotometer was used to monitor the secondary conformation of these two growth hormones after each step of the refolding process. The CD spectra of each refolding stage showed broad absorption in the far-UV range from 210 to 220 nm which represents the
* and n
* transitions of the
-helix conformation and the gradual increase in molar ellipticity [
] absorbance (Figure 2A and B
). A similar
-helix conformation spectrum was also observed for native human growth hormone (data not shown). However, only one
* transition absorption peak was observed from the aromatic ring of tyrosine around 270 nm at the fourth refolding state of both rPaGH and rEaGH (Figure 2C and D
). The subtraction of the first and second refolding stage CD profiles (from 270 to 280 nm) revealed that the proteins had restored the disulfide bond (data not shown).
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SDSPAGE analysis showed that the purities of the refolded rPaGH and rEaGH are 90 and 95%, respectively, by densitometry (Figure 3A). The functional activities of rPaGH and rEaGH that stimulate cell proliferation were analyzed by MTSformazan conversion in ZFL cells. In general, rPaGH is more effective than rEaGH for ZFL cell proliferation. The cell proliferation rates are significantly increased at 1.0 pM and reach to 218 and 158% at 1.0 µM rPaGH and rEaGH, respectively (Figure 3B
).
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Discussion |
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In this study, we developed a process by which the structure and function of recombinant fish growth hormone can be recovered from aggregation form. The aggregated fish rGH may dissolve and unfold in a high-pH buffer and refold towards approximate native form in stepwise refolding buffer. During the refolding process, the refolding intermediates were stable and contained a unique secondary conformation, as determined optically under each refolding buffer. The presence of these stable, structural intermediates indicates that the protein folding pathway is a hierarchical process and the folding intermediates also show a stable helical conformation (Figure 2A and B). The folding intermediates may exist in a molten globule state.
Base on a sequential model, the folding pathway can be expressed as follows:
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Secondary conformation prediction, hydropathy plots (Figure 1) and CD absorptions (Figure 2
) at 222 nm, which is the electron n
* transition of helical conformation at variant folding states, indicate that rPaGH and rEaGH may fold in different paths. The fluctuation of folding intermediates in rPaGH or rEaGH may represent variant interactions between solvent environment and different proteins. Therefore, this would not exclude other possible intermediates under different refolding conditions.
The zipper model (Hong and Schellman, 1992) and the study of Scholtz et al. (Scholtz et al., 1991
) indicate that the initiation of the folding process should be a rate-limiting step. Therefore, a prolonged reaction process may overcome the low probability of initiating folding and increasing the folding ratio. In this study, 72 h of equilibrium dialysis in the initial step is responsible for the successful initiation of refolding.
Stepwise refolding processes are generally applied to refold proteins. However, the selection of buffer compositions used in this study is based on several parameters for fitting the physical chemistry characteristics of rGH. Based on similar selection processes, this approach can be widely adopted for other proteins. The details of our buffer selection are as follows. A low ionic strength of 10 mM TrisHCl was selected for the storage of proteins, because both rPaGH and rEaGH are slightly hydrophilic. pH 8.8 was chosen for the refolding process because recombinant fish growth hormone is more stable at a high pH. The stabilizing reagent and chemical chaperonin, glycerol (5% v/v), was used to facilitate the refolding process (Gekko, 1981; Gekko and Timasheff, 1981
). During the first refolding stage, a dilute denaturing reagent, urea (1 M), serves as a steric blocker to reduce the rotation rate and lower the formation of non-specific hydrogen bonds. A previous study also suggested that dilute urea would block incorrect disulfide bond formation (Lin and Kim, 1989
). The high pH (11) in the first two refolding buffers may limit incorrect disulfide bond formation (the pK value of Cys is 8.5) by the hypothesis of conformation energy minimization. No specific oxidizing agents were used in the folding process, but air oxidation was allowed so as to perform the reaction process gradually.
The expected absorption peak in the range 270280 nm, due to a tertiary side chain interaction of tyrosine and phenylalanine, was indeed observed for the refolded recombinant growth hormones (Figure 2C and D). These indicate that the folding intermediates are retained in a partial folding or in a molten globule state. The CD profiles also indicate that the intermediates are not the combination profiles of the native and denature states.
In this study, the refolded rPaGH and rEaGH could stimulate ZFL cell proliferation. Residual denaturants and reducing agents used for the unfolding and refolding processes sometimes cause toxicity in the cell assays. At a 1.0 µM concentration, rPaGH and rEaGH showed no toxicity at all. This indicates that rPaGH and rEaGH are biologically active proteins (Figure 3B). Moreover, human GH does not support the proliferation of ZFL cells at all, whereas ayu and grouper refolded rGH stimulate the proliferation to a great extent. Interestingly, ayu GH shows more stimulation than grouper GH, as ayu and zebrafish are phylogenetically more related than grouper.
It is worth noting that the folding of intermediates of a protein plays an important role in vivo, as it may lead to the correct folding of a functional protein. We have shown that by manipulating the in vitro solvent environment the major helical conformation of folding intermediates and folded product can be recovered from their insoluble forms and further refolded into functional entities. Although some unexpected modifications may occur during the renaturing process under a high pH environment, it seems that they do not affect the major structural conformation or the bioactivity of the final products. The solvent selection system presented in this study could be widely adopted for use with other non-membrane-spanning proteins of near-neutral hydropathy. This and other work in progress lend credence to such potential. This approach also provides a way to recover serial folding intermediates for protein folding studies.
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Notes |
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Acknowledgments |
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References |
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Received September 11, 2000; revised February 6, 2002; accepted February 9, 2002.