Agrotechnological Research Institute (ATO BV), Bornsesteeg 59, 6708 PD Wageningen, The Netherlands
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Abstract |
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Keywords: collagen-like protein/hydrophilic gelatin/Pichia pastoris/proteolytic stability/synthetic gene
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Introduction |
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Several reports have described the production of recombinant gelatin-like proteins in
Escherichia coli
. Analogously to the natural amino acid sequence of the collagen triple-helix forming domain, synthetic genes are constructed from repeating (GlyXaaYaa)
n
-encoding oligonucleotides, where Xaa and Yaa are often proline (Goldberg et al.1989
; Obrecht et al.1991
; Gardner et al.1993
; Cappello and Ferrari, 1994
). Gene instability problems are commonly observed with such highly repetitive genes (
Capello and Ferrari, 1994
). Also, expression levels usually obtained in
E.coli
are rather low and purification of the intracellularly produced protein can be difficult. Recently, Kajino
et al.
reported the use of
Bacillus brevis
for the expression of gelatin-like proteins (Kajino et al.2000
). They used sequence stretches selected from natural collagen genes and polymerized them to form semi-synthetic gelatin. Prior to their report, we reported the use of the methylotrophic yeast
Pichia pastoris
as a superior host for the secretion of recombinant gelatins having natural amino acid sequences, at up to 14.8 g/l of clarified broth (Werten et al.1999
).
Having established the suitability of P.pastoris for the expression of natural recombinant gelatins, we set out to investigate the possibilities of producing entirely custom-designed gelatins having novel physico-chemical properties. A monomeric gene encoding a highly hydrophilic 9 kDa gelatin was designed such as to allow convenient polymerization into larger multimers. The monomeric gene is much longer than the single oligonucleotide monomers used in the expression of synthetic gelatins in E.coli , mentioned above. This offers more flexibility in the design of the amino acid sequence and a concomitant decrease in the overall repetitiveness of the gene. Here, we describe the high-level secretion of a fully synthetic, highly hydrophilic and non-degraded 36.8 kDa gelatin by P.pastoris and its characterization.
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Materials and methods |
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The monomeric gelatin gene (referred to hereafter as `P' for `polar') was constructed by overlap extension polymerase chain reaction (PCR) (Ho et al.1989
) of long oligonucleotides (underlined in Figure 1A
)
. PCR was performed with a Perkin-Elmer GeneAmp 9700, using the proofreading enzyme
Pwo
DNA polymerase (Eurogentec). The 5' half of the gene was constructed by overlap extension of the first and second oligonucleotides and co-amplified by outer primers directed against nucleotides 126 (sense) and 197174 (antisense). Likewise, the 3' half of the gene was constructed by overlap extension of the third and fourth oligonucleotides and co-amplified by primers directed against nucleotides 174197 (sense) and 363337 (antisense). The resulting PCR products were isolated from an agarose gel and were combined by another overlap extension PCR and co-amplified with the primers directed against nucleotides 126 (sense) and 363337 (antisense). The resulting 0.3 kb PCR fragment was digested with
Xho
I/
Eco
RI and cloned in vector pMTL23 (Chambers et al.1988
) to form vector pMTL23P. The sequence of the gene was verified by automated DNA sequencing of both strands.
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Transformation of P.pastoris
Plasmid pPIC9P4 was linearized with
Sal
I in order to obtain preferentially Mut
+
transformants [i.e. by integration at the
his4
locus rather than the
AOX1
locus and thus allowing normal growth on methanol (Clare et al.1991b
)]. Transformation of
P.pastoris
strain GS115 [
his4
(Cregg et al.1985
)] by electroporation and selection of Mut
+
transformants was as described previously (Werten et al.1999
).
Fermentative production of synthetic gelatin in P.pastoris
Fermentations were performed in 1140 l fermenters (Applikon) in minimal basal salt medium (Invitrogen) supplemented with 0.2% (v/v) PTM
1
trace salts (Invitrogen). Methanol fed-batch fermentations were performed as described previously (Werten et al.1999
), with the exception that no protease-inhibiting supplements such as casamino acids were added and that the pH during methanol fed-batch was maintained at 3.0 for all fermentations.
Small-scale purification of synthetic gelatin by differential acetone precipitation
Differential acetone precipitation was as described previously (Werten et al.1999
). Chilled acetone was added to fermentation supernatant at 40% (v/v), after which endogenous proteins were pelleted by centrifugation. The acetone concentration in the supernatant was then increased to 80% (v/v) and the pellet obtained after centrifugation was washed with 80% acetone and air-dried.
Preparative purification of synthetic gelatin by differential ammonium sulfate precipitation
Preparative purification of synthetic gelatin from fermentation supernatant consisted of twice-repeated ammonium sulfate precipitation at 40% saturation (4°C) and subsequent washing of the precipitate with 60% saturated ammonium sulfate. Depending on the scale of the purification, separation of the precipitate from the liquid was either by centrifugation or by depth filtration using AKS-4 sheets (USF Seitz-Schenk). The protein was subsequently desalted by diafiltration and lyophilized.
Bicinchoninic acid protein assay
A commercially available bicinchoninic acid (BCA) protein assay was used according to the manufacturer's recommendations (Pierce). The reaction was performed at 60°C for 30 min. The calibration curve was prepared gravimetrically from lyophilized, desalted P4 gelatin, purified by differential ammonium sulfate precipitation (purity at least 98%).
SDSPAGE
SDSPAGE (
Laemmli, 1970
) was performed in a Mini-PROTEAN II system (Bio-Rad) under reducing denaturing conditions. Gels consisted of a 5% stacking and a 12.5% separating zone (2.7% cross-linking). Gels were stained using Coomassie PhastGel Blue R-350 (Amersham-Pharmacia Biotech) and were destained by heating in water using a microwave oven, similarly to Faguy
et al.
(Faguy et al.1996
).
Gel filtration chromatography
Protein in 0.1 M sodium chloride was loaded on a 10x250 mm column packed with Superose 12 (Amersham-Pharmacia Biotech). Elution was carried out with 0.1 M sodium chloride at a flow-rate of 0.2 ml/min, collecting 2 ml fractions and monitoring the absorbance at 214 nm.
Mass spectrometry
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed at the Department of Biochemistry, Wageningen University, The Netherlands, using a Voyager DE-RP delayed extraction mass spectrometer (PerSeptive Biosystems). Samples were prepared by the dried droplet method, using sinapinic acid dissolved in 30% (v/v) acetonitrile, 4% (v/v) trifluoroacetic acid as matrix. Measurements were made in the positive, linear mode and the accelerating voltage was 25 000 V. Cytochrome c and bovine serum albumin were used as external calibrants.
Chemical modification of gelatins
Esterification of carboxylic amino acid side chains was adapted from Wilcox (
Wilcox, 1967
). A 100 µg amount of protein was incubated in 500 µl of methanol, 0.1 M hydrochloric acid at 4°C for 72 h. The methanol was exchanged for 1 mM hydrochloric acid by diafiltration in a 3 kDa Microcon (Millipore). Removal of ester groups was performed by incubating the esterified protein in 100 mM TrisHCl, pH 8.8 at 20°C for 72 h.
Hydrazination of carboxylic amino acid side chains was performed according to Matagne
et al.
(
Matagne et al., 1991
). A 50 µg amount of protein was dissolved in 20 µl of 50 mM sodium phosphate buffer, pH 7. After addition of 170 µl of 8 M urea, 1 M hydrazine and 0.1 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, pH 4.5, the mixture was incubated at room temperature for 2 h.
Surface tension measurements
Surface tension at the liquidair interface was measured according to the du Noüy ring method (
Lecomte du Noüy, 1919
) using a Krüss K6 tensiometer. The temperature of the sample vessel was maintained at 20°C. The actual measurements were performed 5 min after lowering of the ring on to the liquid surface, as suggested by Clarkson
et al.
(Clarkson et al.1999
). Bovine serum albumin was used as a reference protein and raw data were corrected for the hydrostatic volume effect according to Harkins and Jordan (
Harkins and Jordan, 1930
).
Circular dichroism spectrometry
Proteins were dissolved in Milli-Q water at 0.1 mg/ml. Measurements were performed at the Department of Biochemistry, Wageningen University, The Netherlands using a Jasco J-715 spectropolarimeter. The pathlength was 0.1 cm and spectra were recorded from 190 to 260 nm at 4°C, using a scanning speed of 20 nm/min at a resolution of 0.1 nm.
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Results |
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The basic structure of natural gelatins consists of repeating GlyXaaYaa triplets, where Xaa and Yaa are often proline and hydroxyproline, respectively (the latter being posttranslationally modified proline). This structure maintains the open, unfolded conformation characteristic of gelatin. Owing to this unfolded conformation, gelatin is fairly hydrophilic because its hydrogen bonds are highly exposed. Furthermore, only a small fraction of the protein is occupied by hydrophobic amino acids such as Trp, Tyr, Phe, Leu, Ile, Val and Met.
Our synthetic P4 gelatin design also provides the (GlyXaaYaa)
n
structure. To increase its hydrophilicity relative to that of natural gelatins, we designed a gelatin without any hydrophobic amino acids other than proline and with a high content of the hydrophilic amino acids asparagine and glutamine (Table I
)
. To illustrate the high hydrophilicity of this synthetic gelatin compared with natural gelatins, the GRAVY values [grand average of hydropathy (
Kyte and Doolittle, 1982
)] of P4, natural recombinant Col3a1 gelatin (Werten et al.1999
) and cattle bone gelatin are indicated in
Table
I
.
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The P4 gene was constructed from four identical P monomers that were designed to have the codon usage of
P.pastoris
highly expressed genes (
Sreekrishna and Kropp, 1996
). The monomeric gene contains restriction sites for
Dra
III and
Van
91I. These enzymes allow the design of mutually complementary, non-palindromic overhangs that enable convenient elongation of the gene by insertional doubling in a fixed orientation (Figure 1
). The process can be repeated until the desired polymer length has been achieved. This modular design offers flexibility in the construction of future gelatins by allowing the combination of different types and lengths of polymerized gelatins via the
Dra
III/
Van
91I sites. The
Xho
I and
Eco
RI sites provided by the sequence allow the direct insertion of the final synthetic gene into
P.pastoris
expression vector pPIC9, resulting in a fusion to the alpha-mating factor prepro secretion signal. Thus, the combined four P modules were cloned into pPIC9 to yield vector pPIC9P4.
Production of synthetic P4 gelatin
Plasmid pPIC9P4 was used to transform
P.pastoris
GS115. Randomly chosen transformants were fermented and culture supernatants harvested throughout the fermentation were subjected to SDSPAGE. The theoretical molecular weight of P4 is 36.8 kDa. Because collagenous proteins migrate in SDSPAGE at an apparent molecular weight ~40% higher than the true molecular weight (Butkowski et al.1982
; Werten et al.1999
), one would expect a band of ~52 kDa. Instead, however, the Coomassie Blue-stained SDSPAGE gel showed a faint blurry band at the top of the separating gel that had a tendency to diffuse from the gel during methanolacetic acid destaining. Migration of the gelatin into the gel was improved by running the gel at 4°C at twice the voltage recommended by the manufacturer of the electrophoresis system (i.e
.
400 instead of 200 V
). Diffusion of the protein from the gel during destaining was reduced by destaining the gel in water heated in a microwave oven [similarly to Faguy
et al.
(Faguy et al.1996
)], rather than performing the common lengthy incubations in methanolacetic acid. Figure 2
, lane 1 shows fermentation supernatant analyzed in this manner. N-Terminal protein sequencing of this band (Sequencing Centre Utrecht, The Netherlands) revealed the expected amino acid sequence (GPPGEPGNPG). There was no indication of incomplete processing of the
-factor derived GluAla repeats by dipeptidylaminopeptidase, such as is occasionally observed when using this prepro sequence for secretion of heterologous proteins (Vedvick et al.1991
; Briand et al.1999
; Werten et al.1999
; Goda et al.2000
).
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We previously found that recombinant gelatins with natural amino acid sequences can also be purified from
P.pastoris
fermentations using differential ammonium sulfate precipitation (unpublished data). Gelatinous proteins precipitate at 40% saturation, whereas endogenous extracellular
P.pastoris
proteins surprisingly do not precipitate at up to 80% saturation. In agreement with this, readily precipitable proteins such as ß-lactoglobulin are rendered virtually unprecipitable upon mixing them with fermentation supernatant. We investigated whether it was possible to purify P4 gelatin by differential ammonium sulfate precipitation. Indeed, Figure 2
, lanes 4 and 5 show that synthetic gelatin is quantitatively precipitated at 40% ammonium sulfate saturation, while no endogenous proteins are visible. Based on amino acid analysis (in triplicate) and subsequent linear least-squares fitting of the observed data, the purity at the protein level was estimated to be >98.1% (±0.7% SD). Two-dimensional electrophoresis followed by silver staining showed virtually no contaminants (not shown). Gelatin yields determined by BCA analysis of ammonium sulfate precipitates of several fermentations [possible only after thorough desalting because ammonium sulfate reduces BCA reactivity (Smith et al.1985
)] were within 0.3 g/l of the values determined by acetone precipitation. This small difference (<10%) is largely due to interference of the BCA assay by the small amount of reducing exopolysaccharides that co-purifies in the acetone precipitation procedure, while exopolysaccharides are virtually eliminated when using ammonium sulfate precipitation (data not shown). Compared with differential acetone precipitation, the overall higher purity obtained and the higher amenability to scale-up render differential ammonium sulfate precipitation the method of choice for preparative purification of P4 gelatin.
Establishing the molecular weight of synthetic P4 gelatin
A possible explanation for the aberrant molecular weight observed in SDSPAGE could be that P4 is glycosylated. N-Linked glycosylation can be ruled out because no susceptible sites are present in the amino acid sequence. However,
P.pastoris
is also able to perform
O
-glycosylation and the structural determinants for such an event are unclear (Duman et al.1998
). To rule out this possibility, periodic acidSchiff staining (Zacharius et al.1969
) and Alcian Blue staining (
Wardi and Michos, 1972
) were performed on ammonium sulfate-purified P4. No glycosylation was observed (not shown).
To determine whether the synthetic gelatins have in fact the correct molecular weight, but merely exhibit aberrant behavior in SDSPAGE, analytical gel filtration chromatography was performed. The Superose 12 column was calibrated with a mixture of natural recombinant gelatin fragments (Werten et al.1999
), giving a series of molecular weights of 53, 42, 28, 16, 12 and 8 kDa. Ammonium sulfate-purified P4 was subjected to gel filtration chromatography. Only one significant peak was observed. N-Terminal protein sequencing of this fraction in solution (Sequencing Centre Utrecht) showed the correct N-terminus for P4. The molecular weight of P4 deduced from the chromatogram was 47 kDa. This is clearly much closer to the theoretical value of 36 kDa than the molecular weight apparent from SDSPAGE, although the deviation is still significant.
Mass spectrometry was used to determine ultimately the molecular weight of P4. Materials purified by both ammonium sulfate precipitation and gel filtration chromatography were analyzed and the results were in good mutual agreement. Figure 3
shows the MALDI-TOF mass spectrum of P4 purified by ammonium sulfate precipitation. The observed molecular weight of 36 835 Da corresponds well with the theoretical value of 36 818 Da. This result shows that the apparent high molecular weight observed in SDSPAGE is indeed the result of aberrant migration behavior. Furthermore, the SDSPAGE and gel filtration chromatography results are confirmed, in that there is no presence of proteolytically degraded fragments.
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In marked contrast with recombinant gelatins having natural sequences, we noticed that highly concentrated solutions of P4 showed essentially no foaming. A direct relationship exists between protein (surface) hydrophobicity, surface tension and foam stability (Horiuchi et al.1978
). Therefore, the surface activity of P4 relative to that of Col3a1 natural recombinant gelatin was determined, using the du Noüy ring method (
Lecomte du Noüy, 1919
). Figure 5
shows that P4 does not show any significant lowering of the surface tension of water at concentrations up to 5% (w/v), whereas Col3a1 already has an effect at 0.01%. Within the range up to 10% of protein, it was not possible to determine the apparent critical micelle concentration (CMC; i.e. the concentration whereby the surface tension curve reaches a plateau phase) for either of the gelatin types. For comparison, bovine serum albumin has an apparent CMC of about 0.003% (Clarkson et al.1999
).
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Discussion |
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Despite the obvious physico-chemical differences, both synthetic P4 and natural recombinant gelatin could be purified from the fermentation broth by both differential acetone precipitation and differential ammonium sulfate precipitation. The universality of the two purification techniques is probably due to the hydrophilicity and unfolded structure of gelatins in general. Especially differential ammonium sulfate precipitation allowed convenient large-scale purification of P4 gelatin to near homogeneity.
Secreted synthetic gelatin was fully intact, as evidenced by SDSPAGE, gel filtration chromatography, N-terminal sequencing and mass spectrometry. This is in contrast with natural recombinant gelatins produced in
P.pastoris
, which were partly degraded (Werten et al.1999
). Apart from the occurrence of some minor background degradation, collagen type I-derived natural recombinant gelatins were cleaved into several major bands by a Kex2-like protease. Cleavage occurred C-terminal of two occurrences of the mono-arginylic sequence MetGlyProArg. We speculated that the amino acids occupying the 2 and 4 positions in this motif (relative to the site of cleavage) were a major factor determining the cleavage efficiency (Werten et al.1999
), which is in accordance with recent data on the substrate specificity of
Saccharomyces cerevisiae
Kex2 (Bevan et al.1998
; Suzuki et al.2000
). It may well be that the above-mentioned minor background degradation represented a limited extent of cleavage at Arg residues having `suboptimal' residues at the 2 and 4 positions. Therefore, in the design of the synthetic gelatin described here, only Lys was used as a basic residue to control the isoelectric point. The finding that secreted P4 was completely intact does indeed suggest a general susceptibility of Arg residues in recombinant gelatins to proteolysis and may thus have implications for the rational design of (partially) unfolded proteins to be expressed extracellularly in
P.pastoris
.
While the molecular weight of P4 as determined by mass spectrometry was in good agreement with the value deduced from the amino acid sequence, the molecular weight apparent from gelatin-calibrated gel filtration chromatography was about 10 kDa higher. Ionic interactions with Superose 12 are negligible in the presence of salt and only small hydrophobic peptides appear to show significant hydrophobic interactions with this matrix (Andersson et al.1985
). It is therefore not very likely that a lower degree of such interactions of P4 relative to natural gelatins causes the seemingly aberrant molecular weight. Possibly the effect is due to an increased hydrodynamic size of P4 relative to natural gelatins, as a result of increased interaction of this highly hydrophilic protein with water.
Natural gelatins migrate about 40% more slowly in SDSPAGE than expected. Several possible explanations for the aberrant migration behavior of gelatins have been suggested (
Furthmayr and Timpl, 1971
; Freytag et al.1979
;
Hayashi and Nagai, 1980
; Noelken et al.1981
; Butkowski et al.1982
). It is most likely not the result of anomalously low SDS binding, but is at least in part due to the low average residue molecular weight of gelatin, resulting in a relatively high number of residues (i.e. molecular length) per unit of molecular weight (Freytag et al.1979
; Noelken et al.1981
; Butkowski et al.1982
). Most other reports on aberrant protein migration rates in SDSPAGE involve highly acidic proteins that show reduced binding of SDS due to electrostatic repulsion by the protein's high negative net charge (
Ohara and Teraoka, 1987
; Matagne et al.1991
; Casarégola et al.1992
; McGrath et al.1992
). SDSPAGE showed that P4 migrates at a highly reduced rate, even much more slowly than natural gelatins. We showed here that esterification of the carboxylic side chains of P4 restores its migration rate roughly to that expected for normal gelatins (i.e. about 40% more slowly than common proteins). In contrast, hydrazination did not affect the migration rate of P4. Hydrazination eliminates the negative charge of the same carboxylic residues as does esterification, but reduces the protein's hydrophobicity whereas esterification increases it. As the binding of SDS to proteins is primarily hydrophobic in nature (
Reynolds and Tanford, 1970
), the extremely slow migration of the highly polar P4 gelatin in SDSPAGE is therefore most likely the result of insufficient SDS binding and a concomitant low negative net charge. The finding that the resolution of the SDSPAGE was improved by increasing the field strength to twice that recommended by the manufacturer of the electrophoresis system indicates that the higher field strength aids protein migration in overcoming diffusive forces.
Surface activity is a major determinant in a protein's function as a protective colloid (e.g. in photographic emulsions). Solutions of P4 showed essentially no foaming and tensiometric analysis of solutions of P4 in water showed only negligible surface activity. P4 thus represents a novel hydrocolloid combining some of the characteristics unique to gelatins and a low surface activity commonly expected only for the most hydrophilic polysaccharide hydrocolloids.
We previously showed that non-hydroxylated, natural recombinant gelatins do not show triple-helical structure in circular dichroism spectrometry (Werten et al.1999
). It is a well-established fact that hydroxyproline residues play a crucial role in the stabilization of the collagen triple helix. This role is easily recognized when examining the amino acid compositions and thermal stabilities of natural collagens from different species (
Privalov, 1982
). X-ray crystallography showed that the triple helix is surrounded by a cylinder of hydration (Bella et al.1994
). Although recently questioned (Holmgren et al.1999
; Nagarajan et al.1999
), the role of hydroxyproline in the stabilization of the triple helix is generally attributed to its hydrogen bonding with this water network (
Brodsky and Shah, 1995
). In view of the high polarity of P4, we considered it prudent to investigate its conformation using circular dichroism spectrometry. No triple helical structure was observed at 4°C and P4 gelatin is thus an essentially non-gelling gelatin. Non-gelling gelatins permit novel applications such as low-temperature silver halide crystallization in the preparation of photographic emulsions (de Wolf et al.2000
). Comparison of the circular dichroism spectrum of P4 with that of Col3a1 natural recombinant gelatin showed that the mean residue ellipticity of P4 at the discriminating wavelength of 221 nm was actually lower than that of Col3a1. Although the latter is essentially in a random coil conformation, the higher hydrophilicity of P4 probably reduces minor intramolecular and intermolecular interactions, thereby resulting in a slightly lower ellipticity.
Current research is directed towards the production of other synthetic gelatins with distinct functionalities and combining them to form chimeric tailor-made biopolymers.
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Notes |
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2 Present address: NIZO Food Research, Kernhemseweg 2, 6718 ZB Ede, The Netherlands
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Acknowledgments |
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Received December 11, 2000; accepted April 23, 2001.