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Production of two aprotinin variants in Hansenula polymorpha
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Process Biochemistry, Vol. 31, No. 7, pp. 679-689, 1996 Copyright © 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0032-9592/96 $15.00 + 0.00
ELSEVIER
PI 1:S0032-9592
(96)00018-0
Production of Two Aprotinin Variants in
Hansenula polymorpha Christian Z u r e k , a E d w a r d Kubis, a P e t e r Keup, a Dietrich H6rlein, b Jiirgen Beunink, b J6rg T h 6 m m e s , c M a r i a - R e g i n a Kula, c Cornelis P. H o l l e n b e r g a & G e r d Gellissen a * "Rhein Biotech GmbH, Eichsfelder Strasse 11, 40595 Diisseldorf, Germany b Bayer AG, Pharma-Biotechnologie, 42096 Wuppertal, Germany c Institut for Enzymetechnologie, Heinrich-Heine-Universit~it Dtisseldorf, 52404 Jiilich, Germany a Institut for Mikrobiologie, Heinrich-Heine-Universit~it Diisseldorf, 40228 D~isseldorf, Germany (Received 9 January 1996; accepted 10 February 1996)
DNA-sequences coding for two DesPro(2) aprotinin variants were expressed in the methylotrophic yeast Hansenula polymorpha from a strong inducible promoter element derived from the MOX gene, a key gene of methanol metabolism. For secretion the coding sequences were fused to the KEX2 recognition site of the S. cerevisiae-derived MF~ 1 preproleader sequence. Correct processing of the precursor molecules and efficient secretion of the mature proteins was observed. A pH/pO2-controlled C-source feeding mode was applied for ferrnentations on a 10 litre scale. In cultures of a transformant strain harbouring 20 copies of the aprotinin expression cassette a yield of 350 mg/litre could be obtained. The secreted recombinant products were purified in a simple two-step isolation procedure employing an expanded bed adsorption as an initial purification step. Copyright © 1996 Elsevier Science Ltd
INTRODUCTION
tial as a therapeutic and diagnostic compound 6'7 forced the development of effective recombinant expression systems. Consequently, their establishment has been a major target of genetic engineering during the recent past after the respective gene sequences became available. A preferred production system is based on the yeast Saccharomyces cerevisiae 8-1° fusing the aprotinin sequence to a prepro-~-factor leader segment 11 for product secretion. High levels of incorrectly processed molecules have been encountered in a Pichia pastoris system due to steric features of the newly created fusion siteJ 2 Improvements have been described for S. cerevisiae-derived products deleting the proline residue in position 2 of the aprotinin chainJ °
The bovine pancreatic trypsin inhibitor aprotinin is a small protein of 6.5kDa and was initially isolated by Kunitz and Northrop. ~ The very basic (pI 10.5) single-stranded polypeptide consists of 58 amino acids cross-linked by three disulphide bridges. 2 It is found in a variety of bovine organs including liver and lung. 3 The natural aprotinin inhibits a range of proteases; its specificity can be modified by amino acid substitution within the reactive site. 4"5 The natural isolate has recently obtained FDA approval as a therapeutic. The high poten-
*To whom correspondence should be addressed. 679
680
C. Zurek et ai.
The methylotrophic yeast Hansenula polymorpha has been developed as an expression system for heterologous proteins. 13-~8 As a facultative methylotroph this yeast species is able to use methanol as the sole energy and carbon source; other possible carbon sources are glucose or glycerol. Upon addition of methanol into culture media or in media supplemented with glycerol in low concentrations, key enzymes of the methanol metabolism are strongly produced. The strong inducible promoter elements of the cloned key enzyme genes for the methanol oxidase (MOX) 19 and the formate dehydrogenase (FMD) 2° are used as components for heterologous gene expression providing a production process in recombinant strains which can be induced by compounds of a culture medium. After uptake, the heterologous DNA is stably integrated into the host's genome. Transformations result in a variety of strains harbouring a varying copy number of the integrated DNA in a head-to-tail arrangement. This stable multimeric integration of expression cassettes makes Hansenula polymorpha an ideal host for a genedosage dependent synthesis of foreign proteins. 16-19 As in other yeast expression systems a range of heterologous leader sequence from animal and yeast sources can be used for export targeting, among others the commonly used S. cerevisiae-derived MFo~I leader. 21-24 Purification of secreted proteins from whole fermentation broth demands a clarification step prior to further processing of the compound of interest. The solid-liquid separation step usually involves centrifugation or filtration followed by a concentration of the clarified liquid prior to subsequent purification procedures. Possible approaches to minimise product losses and time requirements of the early clarification and concentration steps are methods which integrate clarification and concentration into a single initial purification step. As such, fluidized bed adsorption has successfully been applied as a real chromatographic purification starting from unclarified broth. 25-3° In the present study we describe the engineering of recombinant H. polymorpha strains secreting two variants of aprotinin, namely the DesPro(2)-Lysl5 and the DesPro(2)-Argl5 aprotinin, and a rapid purification procedure of the secreted products from the culture broth of the engineered strains using
a fluidized bed adsorption as the initial purification step.
MATERIALS AND METHODS Strains and media
Plasmid constructs were propagated in E. coli strain HB10131 supplementing the media with chloramphenicol (30/~g/ml) when required for selection of transformants. The uracil-auxotroph mutant H. polymorpha strain RB11 deficient in oritidine-5'-phosphate decarboxylase (ura3) generated by the standard ethylmethane sulphate method served as standard hosts for the integration of the various plasmids constructed during the course of the present study. The strain was transformed as previously described. 32 In the case of supertransformation, transformation mixtures were plated on YNBplates containing phleomycin [700 ~,g phleomycin (Cayla) in 400/d], applied to the surface of hardened 20 ml agar platesY The antibiotic is applied after transformation allowing growth for about 4 h in advance. Positives were identified by culturing yeast clones under conditions of MOX-promoter derepression (see later) and determining the presence of aprotinin in the supernatants of the cultured cells by an ELISA using calibrated aprotinin samples for standardisation. A monoclonal antibody and aprotinin standards were kindly provided by Bayer AG Wuppertal. Test tubes (3 ml) and shake-flask (50 ml) cultures were grown in YNB [0.67% (w/v) Difco YNB] at 37°C supplemented with 1% (w/v) glycerol as carbon source for culture growth and at lower levels for heterologous gene expression (MOX-promoter derepression; see Introduction). When recombinant strains were grown at 101itre scale, a Biostat ER-10 fermenter (Braun, Melsungen) was charged with 7 litre of a synthetic medium as previously described, is supplemented with 0.2% (w/v) pepton 190, 0.3% (w/v) pepton 5 and 1.5% (w/v) glycerol. A 10% (v/v) Struktol J673 solution (Sichler, Germany) served for foam reduction. After steam sterilisation the medium was inoculated with 1 litre of an overnight culture of the recombinant strain grown in shake-fasks as described above, pH was adjusted with 30% phosphoric acid; base solution was 15% NH3 (w/v) and 50% glycerol (w/v). Glycerol was
Aprotinin variants in Hansenulapolymorpha
added as 50% (w/v) solution under pO2 control and via the base solution. During the consecutive last phase of fermentation a glycerol/methanol mixture (20:80) was fed in pulses for promoter induction. In the initial phase glycerol was added in pH-controlled pulses to maintain a concentration of 1-2% (w/ v). Then after 35 h upon consumption of glycerol a pOE-Controlled feed was initiated to maintain the glycerol concentrations between 0 and 0.2% (w/v) for the next 25 h. This was found to be optimal for promoter depression. During the ultimate induction phase C-source pulses provided glycerol concentrations between 0.0 and 0.2 and methanol concentrations between 0.2 and 1%. The culture conditions were maintained as follows: pH 4.5, 30°C, aeration rate 10 litre/min; stirrer speed 500 rpm. Fermentation on a 21itre scale was performed in a Biostat B fermenter (Braun, Melsungen, Germany) using the synthetic medium and conditions described above. Process parameters were controlled using the MFCS software (micro-multi control fermentersystem) supplied by the company. Glycerol and methanol concentration of fermenter samples were determined by gas chromatography (see below), aprotinin content by HPLC analysis34 and by the methods described before.
Isolation and analysis of nucleic acids Plasmid DNA was prepared from E. coli cultures by the alkaline extraction methodY Total yeast DNA was isolated from 50 ml cultures grown in YNB supplemented with 2% glucose according to Sherman et al. 36 DNA fragment separation and Southern analysis followed standard protocols. 37 Restriction enzymes, ligases and DNA-modifying enzymes were obtained from Boehringer Mannheim and employed according to the manufacturer's instructions. Gas chromatography Gas chromatographic quantification of glycerol and methanol was carried out on a Shimadzu GC-14A equipped with an integrator C-R4AX (Shimadzu), a SPBTM-5 column, length 15 m, thickness of layer 1-5 #m, ID 0.53 mm (Supelco) and a FI detector. Aliquots of centrifuged and sterile-filtered fermentation samples were diluted as 50 pl into 1 ml of 0.01% (v/v) 1-butanol. For separation of samples (sample volume
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1 #1) the column was heated to an initial temperature of 100°C increasing to 250°C at a 15°C/min rate. Injector temperature: 320°C; detector temperature: 320°C. Helium served as carrier gas. A solution of 5 g/litre (w/v) glycerol and 1% (v/v) methanol were used as standards. HPLC analysis Centrifuged and sterile-filtered fermentation samples were diluted as 50 pl into 1 ml of a degassed buffer A [5% (v/v) CH3CN; 0.1% (v/v) TFA; 250 mM NaC104]. Aliquots (100 #l) of these samples were analysed on a Shimadzu CR4AX chromatopac equipped with a Controller 421A, a solvent delivery module 114M, a variable wavelength detector 165 adjusted to 214 nm, and an Altex 210 A valve (all from Beckman). Samples were separated on a 5 #m Spherisorb C8 column (20x4.6mm; PhaseSeparation) and eluted with a mixture of buffer A and a buffer B [80% (v/v) CH3CN; 0.09% (v/ v) trifluoroacetic acid (TFA), the percentage of buffer B increasing from an initial 10% to 35% at 2% min rate]. The flow rate was adjusted to 1 ml/min. Aprotinin content was determined by profile comparison with calibrated aprotinin standards.
Protein analysis Secreted proteins were separated on 15% SDS gels according to Laemmli38 and visualised by Coomassie staining. The N-terminus of aprotinin isolates was determined by automated liquid-phase sequence determination on a pulse liquid-phase peptide sequenator (Applied Biosystem 477A). Aprotinin purification For aprotinin purification 3200 ml of the culture broth (cell mass 100 g dry weight/litre) was diluted 1:1 with deionised water to 25 mS/cm conductivity and adjusted to pH 3.5. 300 ml (settled bed height 15 cm) of Streamline-SP cation exchanger (Pharmacia, Freiburg, Germany) were filled into a commercially available 5 cm diameter column for expanded bed adsorption (Streamline 50, Pharmacia, Freiburg, Germany). The adsorbent was fluidised at a linear flow rate of 5 cm/min and equilibrated with 10 column volumes of 20mM sodium citrate buffer pH 3.5 resulting in a three-fold bed expansion. Diluted broth (6400 ml) was applied at 5 cm/min, unbound proteins and resi-
682
C. Zurek et al.
dual biomass were subsequently washed from the expanded bed with equilibrating buffer. The bed was then allowed to settle and two elution procedures were performed in a fixed bed mode. In a first step employing 20 m u sodium citrate with 0"5 U sodium chloride contaminating proteins were desorbed. Crude aprotinin was eluted using 20 m u sodium citrate with 0.9 M sodium chloride. The matrix was regenerated with 2 M sodium hydroxide in a fixed bed mode as well. The aprotinin-containing eluate was supplemented with 0.1% (v/v) of TFA and applied to a RP 18 Lichroprep HPLC column (Merck, Darmstadt) at 2 cm/min without further pre-treatment. Pure aprotinin was eluted using a gradient from 0.1% TFA in aqueous solution to 50% of 0.1% (v/v) TFA in 40% (v/v) isopropanol. The aprotinin-containing fractions were desalted by gel filtration on a Sephadex G25 column (Pharmacia, Freiburg, Germany) and freeze-dried.
medium to obtain mitotically stable transformants bearing the integrated foreign DNA. In the case of phleomycin at a concentration of 35#g/ml. Out of several hundred clones
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RESULTS
(B) Plasmid construction A 0.5 kb DNA fragment containing a synthesised MF~l-leader/aprotinin fusion (desPro(2)Argl5) was cloned into the multiple cloning site of plasmid pRBMOX located between a MOXpromoter and a MOX-terminator element thus constituting an expression cassette for the secretion of aprotinin. The construction results in the plasmid pRBMOXApro (see Fig. 1A). For construction of the plasmid pRBMAproPhle a phleomycin resistance gene was inserted into this vector (Fig. 1B). For generation of the respective plasmid for the expression of DesPro(2)-Lysl5 the fusion fragment was subcloned into a M13 vector and mutagenised by site directed mutagenesis. The successful mutagenesis was controlled by sequencing, and the newly obtained fragments were inserted into the basic expression vectors as described before. Generation of aprotinin-secreting
H. polymorpha strains After rounds of transformation with the two pRBMOXApro constructs described before (for details see the Materials and Methods section), colonies secreting aprotinin were isolated and sequentially grown for 70 generations in selective (YNB) and for two passages in rich (YPD)
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Fig. 1. Physical maps of the expression vectors for aprotinin used in this study. The various MFctl/aprotinin fusions were inserted as EcoRI/Bg/II fragments into the multiple cloning site of the H. polymorpha expression/ integration vector pRBMOX separating a MOX-promotor (MOX-P.) and a MOX-terminator (MOX-T.) sequence. The plasmid contains the following additional components: o r / ( O R I ) and a chloramphenicol resistance (cam) gene for selection and propagation in E. coli and a Hansenula autonomously replicating sequence (HARS1) and a URA3 gene (URA3) for propagation and selection in the uracil-auxotrophic H. polymorpha host strain RBll. The resulting plasmid is pRBMOXApro (A). For retransformarion a phleomycin resistance gene obtained from the plasmid pUT332 (CAYLA) was inserted to result in the plasmid pRBMAproPhle (B).
Aprotinin variants in Hansenula polymorpha
obtained examples representative for the different constructs were selected for a more detailed analysis. In preliminary expression studies in fermentations at a 50 ml scale clones were grown in 2% glycerol, secretion of aprotinin was monitored after 65 h ranging from 5 to 20 mg/litre. Examination of genomic DNA by Southern hybridisation identified several strains with different copy numbers of the integrated expression cassette. To estimate the copy number, DNA was restricted with SalI/XbaI, transferred to nitrocellulose filter, and hybridised to a 32p-labelled MOX-promoter probe. The signal intensity of calibrated DNA dilutions could be compared to that of the intrinsic single copy control derived from the host MOX gene and provided an estimation of heterologous gene content. The copy number was determined as a range from 1 to 16 copies in the various strains analysed. In case of a DesPro(2)-Argl5 strain a particular isolate bearing 16 copies of the heterologous expression cassette was retransformed with plasmid pRBMAproPhle and transformants were selected according to the newly introduced resistance. In the example documented in Fig. 2 the selected strain 23-3 is analysed and identified as having acquired two additional copies of the aprotinin expression cassette. In the case of DesPro(2)-Lysl5 variant transformants harbouring a gene dosage of between one and 14 copies could be identified (see Table 1). The integrated DNA was found to be mitotically stable after passaging for more than 80 generations (data not shown). For further studies we focused on the analysis of a single transformant of each construct. The selected strains habour 18 copies (supertransformant of the DesPro(2)-Argl5 type) and 14 copies (DesPro(2)-Lysl5-type), respectively (Fig. 2 and Table 1). The supertransformant expressing the DesPro(2)-Argl5 variant is now designated as strain I, the transformant for the expression of the alternative DesPro(2)-Lysl5 aprotinin as strain II. Expression and properties of aprotinin secreted from strains I and II In a comparative analysis the two strains were cultivated at a 2 litre scale as described in the Materials and Methods section. After 26h under MOX-promoter derepression culture supernatants were harvested and analysed for
683
a
b
O Fig. 2. Copy number determination of the heterologous DNA in recombinant strains. Genomic DNA from a selected retransformed strain (strain I) was digested with SalI/Xba I. Standardised dilutions of the various restriction samples were separated through 0.8% agarose gels, transferred to nitrocellulose filters and hybridized to a 32p-labelled MOX-promotor fragment, comparing the intensity of the resulting hybridisation signals derived from the heterologous fusions with that of the single copy MOX gene. The strain was estimated to contain 16 copies derived from the first transformation with pRBMOXApro and two further copies from the retransformation with pRBMAproPhle. The faint upper signal corresponds to the intrinsic single copy MOX gene; the strongest signal in the middle position to the heterologous fusion resulting from the transformation with pRBMOXApro and that in the lower position to that from retransformation with pRBMAproPhle. (a) Size marker (EcoRI/HindlII digest of 2 DNA). (b) Sal/I/Xba I digest of genomic DNA from strain I (undiluted, dilutions 1:2; 1:4; 1:8; 1:16 and 1:32; from left to right).
Table 1. Properties of selected aprotinin-secreting strains
Strain
Aprotinin variant
Copy number
Productivity Correctly (mg/litre) processed product
(%)
Strain I Strain II
DesPro(2)Argl5 DesPro(2)Lysl5
16 + 2
350
82
14
270
80
C. Zurek et al.
684
aprotinin content. The highest accumulation of aprotinin was observed in the culture of strain I and determined as 150 mg/litre, whereas strain II produced 100 mg/litre, respectively, roughly reflecting the difference in aprotinin gene dosage. Nevertheless, all constructs provide for an efficient secretion confirming that both MF~I/ aprotinin preprostructures are recognised by the secretory system of the methylotrophic host. A comparative HPLC-analysis revealed that the secreted aprotinin consists of a mixture of different species. In all cases the correctly processed full length form represents the majority product in the medium ranging from 80 (strain II) to 82% (strain I) of the overall aprotinin yield (Table 1). Determination of N-terminal amino acid sequences revealed different N-terminal extensions with a main peak harbouring aprotinin of a correctly processed N-terminus (data not shown; see also analysis of purified aprotinin from a 10 litre fermentation).
of the substrate, glycerol was kept at a low level with a pO2-controlled feed to promote heterologous gene expression (condition of MOX-promoter derepression). After a total fermentation time of 50-70h a feed of six consecutive pulsed additions of a methanol/ glycerol mixture (80% methanol, 20% glycerol) for MOX-promoter induction was initiated. Details of the fermentation conditions are given in the Materials and Methods section and in the legend of Fig. 3. After a total fermentation time of 80-100 h, the aprotinin content of the supernatant was analysed as 320mg/litre. The productivity of strain II could be optimised to 270mg/litre applying a similar fermentation regimen (Table 1). Purification of aprotinin accumulated in the culture medium
The purification of aprotinin in a simple twostep procedure was made possible by the fact that only very low levels of contaminating secretory proteins from the H. polymorpha host are present in the culture broth. From the beginning a high product titre simplified the chromatographic purification. Application of the whole culture broth to the cell matrix necessitated a 1:1 dilution of the broth. In this way the conductivity of the medium was lowered to a value where adsorption of aprotinin to the ion
Fermentation studies at 10 litre scale
Productivity of the two strains was further analysed in fermentation runs at 10 litre scale. In the analysis documented in Fig. 3 strain I was cultured following a two-carbon source mode. The cells were first grown in 1-2% glycerol to a dry weight of 30 g/litre in a pHcontrolled feed mode. Then upon consumption
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Fig. 3. Fermentation of strain 1 at a 10 litre scale. A fermentation run was carried out as described in the Materials & Methods section. The fermentation started with 2% glycerol at the beginning and a pH-stat-glycerol feeding mode during the initial growth phase of the culture. After 35 h and the consumption of the carbon source a pO2-controlled feed was initiated resulting in glycerol concentration between 0-0 and 0"2% (w/v) (derepression of the MOX-promoter). After 60 h a methanol/glycerol mixture was fed in six consecutive pulses. Timed fermentation samples were taken and analysed for aprotinin content in the supernatant and other fermentation parameters. After a fermentation time of 85 h the culture broth was harvested and aprotinin was purified from the supernatant.
Aprotinin variants in Hansenula polymorpha
exchanger was favoured; furthermore the viscosity of the broth was reduced, such that the bed did not expand beyond the upper end of the column when being fluidized with the bio~nass-containing sample. The diluted culture broth had a biomass content of 5% dry weight thus being within the range recommended. 25 During adsorption total aprotinin was captured ,)n the ion exchanger. The two-step elution ~rotocol produced 3.8-fold purified and sevenfold concentrated aprotinin at a 68% yield. The clarification of the cell-containing broth was ~atisfactorily achieved in addition to the chromatographic success. Therefore, after addition of 0.1% TFA the eluate was applied to the HPLC column without further treatment. The ~eparation of incorrectly processed aprotinin ~vas accomplished in the RP-HPLC step. HPLC, SDS-PAGE and N-terminal sequencing confirmed the fidelity and homogeneity of the recovered isolate (Figs 4 and 5). The results of the purification are summarised in Table 2.
DISCUSSION
Fed-batch fermentations of particular H. polymorpha strains described in this report followed a two-carbon source mode adding suitable amounts of glycerol and methanol to the broth. Feeding of these two compounds followed a regimen which provides an optimal growth during the initial growth phase and which achieves high product yields during a two-phase production by first derepressing and then inducing the MOX-promoter used for heterologous gene expression. During the initial growth phase a high glycerol content of 1-2% was maintained by a feeding mode coupled to base consumption. In the following derepression phase the glycerol was added by a p O z - c o n trolled feeding mode. Similar strategies have been described using pO2, pH or product concentrations for 'indirect' feed-back c o n t r o l . 39-42 This results in a simple process which is relatively unsusceptable to potential interferences. The methylotrophic yeast H. polymorpha exhibits favourable properties for heterologous gene expression. One major advantage is the potential of this microorganism to secrete large amounts of protein products into the medium. 15-~8 As unicellular eukaryotic organisms, yeasts
685
share to a large extent the basic characteristics of the secretory pathway ubiquitously found in organisms ranging from fungi to mammalians. As a consequence, a primarly translation product can correctly be processes irrespective of the source of a leader sequence used for secretion. 21-24"43 Such a leader may comprise a short 20 amino acid sequence targeting the polypeptide to the endoplasmatic reticulum (ER) and which is cleaved off upon entry into this compartment by a signal peptidase. 44"45 Alternatively, it may consist of a preprosegment. For processing of the latter further proteolytic cleavage of the imported pro-polypeptide is required by a dibasic protease of the Golgi apparatus equivalent to the S. cerevisiae endopeptidase F (gene product of the KEX2 gene).4('
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Fig. 4. HPLC-analysis of aprotinin samples derived from the fermentation described in Fig. 3. HPLC conditions are as described in the Materials and Methods section. (A) Supernatant of the culture broth. The two arrows indicate fractions containing incorrectly processed or degraded aprotionin molecules. (B) Purified aprotinin obtained after the final purification step.
C. Zurek et al.
686
1
2
3
4
5
i I
,q
Fig. 5. SDS-PAGE analysis of aprotinin samples. Denatured protein samples were separated through 15% SDS-PAGE gels and visualised by silver staining. Lane l, Pharmacia low molecular weight marker corresponding o 17.2, 14.6, 8.2, 6.4 and 2.5KDa, respectively; lane 2, supernatant from the fermentations of strain I (see Fig. 3); lane 3, crude aprotinin after the first purification step (0"9 M NaCI eluate from the Streamline SP column); lanes 4 and 5 purified aprotinin samples after the first and the second reversed phase chromatography, respectively.
A variety of leader sequences has been employed in yeast expression systems inclusive of H. polymorpha following the general design of the two basic types, many of them originating from a heterologous source. 21-25"47 However, not all heterologous leader sequences function in yeasts. For instance, those of leech echistatin and human tissue plasminogen activator do not direct secretion in S. cerevisiae.48"49 Furthermore, correct processing might be hampered by structural or steric features of the newly created fusion. Examples for such heterogeneity are available for both basic leader types, among others for aprotinin produced in the methylotrophic yeast P. pastoris. 12 Correct processing can be predicted to some extent by empirical rules. 5° This prediction suggested the deletion of the proline residue in position 2 to create a processing site more suited for KEX2 processing. In the study presented here we employed the Table 2. Summary of the aprotinin purification protocol
Sample
Harvest Streamline SP eluate (0-9 M NaC1) RP 18 HPLC Sephadex G25
Aprotinin concentration (mg/litre )
Purification (fold)
YieM (%)
202 1412
1 3.8
100 76
719 543
5.45 5.5
36 35
preprosegment of MF~I which seemed more suitable to us for direct secretion of smaller products like aprotinin from a H. polymorpha host and which has successfully been applied for the secretion of hirudin from this microorganism 23'24 and for export of aprotinin from S. cerevisiae. Closer inspection and comparison of the secreted products revealed that expressions of both preproaprotinin fusion result in a high share of correctly processed mature protein, contrasting with the situation in P pastor& where an aprotinin sequence including the proline was expressed. Aprotinin species with N-terminal-amino acid extensions make up 15% of the overall aprotinin yield demonstrating that the N-terminal modification has led to a considerable improvement. Secretion provides an attractive mechanism for product recovery without having to break open cells, especially since the methylotrophic host secretes genuine polypeptides in low abundance only. Accordingly, a relatively simple combination of chromatographic procedures can be employed for the purification of secreted aprotinin mainly to exclude incorrectly processed molecules from the desired isolate. The classical strategy used for the purification of recombinant aprotinin is based on an initial clarification of the growth medium, a cation exchange chromatography or affinity chromatography as first enrichment and a final purification by reversed phase HPLC. After desalting by gel filtration and freeze-drying, a pure and correctly processed product is obtained. 5j'52 To simplify the purification process the first three steps were combined in the present study by an expanded bed adsorption employing a commercially available cation exchange material suitable for expanded bed adsorption (Streamline SP). The resulting eluate was found to be ready for direct application to the subsequent purification. The purified aprotinin appeared to be homogeneous and correctly processed. The engineered H. polymorpha strains further attests to the potential of this system for a commercially competitive production processes. The relatively poor productivity of the two strains used in this study is probably the result of the expression cassettes being present in low copy numbers. In other recent examples H. polymorpha strains have been obtained bearing 40 copies of a heterogeneous gene or more which
Aprotinin variants in Hansenula polymorpha
secrete a recombinant product in the gram range. Since the production capabilities for a given protein is obviously correlated to some extent to the dosage of the respective gene in the expressing host it appears likely that further high-copy transformants can be selected for both strain types exhibiting higher productivities. On the other hand, the produced aprotinin might impose a physiological stress on the yeast restricting transformants to a gene dosage and a productivity as observed in the described examples. H. polymorpha provides several advantages as production organism for a compound considered for administration to humans. The mitotically stable introduction of foreign genes creates strains suited for a reproducible fermentation process under non-selective conditions. In risk assessment studies the ecological safety of aprotinin-secreting strains has been s h o w n . 53'54 The characteristics and the performance of the aprotinin-producing strains described in this publication attest together with previous examples TM that the H. polymorpha expression system meets the general demands and prerequisites for industrial application where a system of choice should produce an optimal amount of authentic bioactive material in a reproducible and stable production process. ACKNOWLEDGEMENTS
The authors gratefully acknowledge the financial and logistic support by the Bayer AG, Wuppertal. We thank Mr K.-P. M~ider (Bayer AG) and J. D0rkop (Rhein Biotech GmbH) for their excellent technical assistance and D. Kinzelt, lET, Heinrich-Heine Universit~it Diisseldorf for determination of amino acid sequences. REFERENCES 1. Kunitz, M. & Northrop, J. H., Isolation from beef pancreas of crystalline trypsinogen, trypsin inhibitor, and an inhibitor trypsin compound. J. Gen. Physiol., 19 (1936) 991-1007. 2. Anderer, F. A. & HOrnle, S., The disulphide linkages in kallikrein inactivator of bovine lung. J. Biol. Chem., 241 (1966) 1568-72. 3. Fritz, H. & Wunderer, G., Biochemistry and applications of aprotinin, the kallikrein inhibitor from bovine organs. Arzneim. Forsch./Drug Res., 33 (1983) 479-94. 4. Dietl, T., Huber, C., Geiger, R., Iwanaga, S. & Fritz,
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H., Inhibition of porcine glandular kallikreins by structurally homologous proteinase inhibitors of the Kunitz (Trasylol) type. Significance of the basic nature of amino acid residues in subsite position for kallikrein inhibition. Hoppe-Seyler's Z. Physiol. Chem., 360 (1979) 67-71. 5. Wenzel, H. R., Beckmann, J., Mehlich, A., Schnabel, E. & Tschesche, H., Semisynthetic conversion of the bovine trypsin (Kunitz) into an efficient leukocyteelastase inhibitor by specific valine for lysine substitution in the reactive site. In Chemistry of Peptides and Proteins, Vol. 3, ed. W. Voelter, E. Bayer, Y. A. Ovchimnikov & V. T. Ivanov. DeGruyter, Berlin, 1986, pp. 105-17. 6. Hewlett, G., Aprotinin-Funktion und Wirkung in der medizinishcen Forschung und Entwicklung. Bibengineering, 5/89 (1989) 40-2. 7. Auerswald, E.-A., H6rlein, D., Reinhardt, G., Schr6der, W. & Schnabel, E., Varianten des pankreatischen Rinder-Trypsininhibitors, deren Herstellung und Verwendung (1988) EP 0307 592. 8. Tschesche, H., Beckman, J., Mehlich, A., Schnabel, E., Truscheit, E. & Wenzel, H. R., Semisynthetic engineering of proteinase inhibitor homologues. Biochim. Biophys. Acta, 913 (1988) 97-101. 9. Norris, K., Norris, F., Bjorn, E. S., Diers, I. & Petersen, L. C., Aprotinin and aprotinin analogues expressed in yeast. Biol. Chem. Hoppe-Seyler, 371 (1990) 37-42. 10. Ebbers, J., H6rlein, D., Schedel, M. & Das, R., Rekombinante Aprotinin Varianten - - Gentechnisches Verfahren zur mikrobiellen Herstellung von homogen prozessierten Aprotinin-Varianten sowie die therapeutische Anwendung derselben, (1991) patent application DE 3930522 A 1. 11. Brake, A. J., Merryweather, J. P., Coit, D. G., Heberlein, U. A., Masiarz, F. R., Mullenbach, G. T., Urdea, M. S., Valenzuela, P. & Barr, P. J., 7-Factor-directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae. Proc. Natl Acad. Sci. USA, 81 (1984) 4612-6. 12. Vedvick, T., Buckholz, R. G., Engel, M., Urcan, M., Kinney, J., Provow, S., Siegel, R. S. & Thili, G. P., High-level secretion of biologically active aprotinin from the yeast Pichia pastoris. J. Ind. Microbiol., 7 (1991) 197-202. 13. Roggenkamp, R. O., Hansen, H., Eckart, M., Janowicz, Z. A. & Hollenberg, C. P., Transformation of the methylotrophic yeast Hansenula polymorpha by autonomous replication and integration vectors. Mol. Gen. Genet., 202 (1986) 302-8. 14. Gellissen, G., Strasser, A. W. M., Meiber, K., Merckelbach, A., Weydemann, U., Keup, P., Dahlems, U., Piontek, M., Hollenberg, C. P. & Janowicz, Z. A., Die methylotrophe Hefe Hansenula polymorpha als Expressionssystem f/Jr heterologe Proteine. Bioengineering, 5/90 (1990) 20-6. 15. Gellissen, G., Weydemann, U., Strasser, A. W. M., Piontek, M., Janowicz, Z. A. & Hollenberg, C. P., Progress in developing methylotrophic yeasts as expression systems. TIBTECH, 10 (1992) 413-17. 16. Gellissen, G., Janowicz, Z. A., Weydemann, U., Melber, K., Strasser, A. W. M. & Hollenberg, C. P., High-level expression of foreign genes in Hansenula polymorpha. Biotech Adv., 10 (1992) 179-89. 17. Gellissen, G., Heterologous gene expression in C~ compound-utilizing yeasts. In Recombinant Microbes for Industrial and Agricultural Applications, eds Y.
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Murooka & T. Imanaka. Marcel Dekker, New York, 1994, pp. 787-96. 18. Gellissen, G., Hollenberg, C. P. & Janowicz, Z. A., Gene expression in methylotrophic yeasts. In Gene Expression in Recombinant Microorganisms, ed. A. Smith. Marcel Dekker, New York, 1994, pp. 195-239. 19. Ledeboer, A. M., Edens, L., Maat, J., Visser, C., Bos, J., Verrips, C. T., Janowicz, Z. A., Eckart, M., Roggenkamp, R. O. & Hollenberg, C. P., Molecular cloning and characterization of a gene coding for methanol oxidase in Hansenula polymorpha. Nucleic Acid Res., 13 (1985) 3063-82. 20. Hollenberg, C. P. & Janowicz, Z. A., DNA molecules coding for FMDH control regions and structured gene for a protein having FMDH activity and their uses, (1988) European patent application EPA 0299108. 21. Gellissen, G., Melber, K., Janowicz, Z. A., Dahlems, U., Weydemann, U., Piontek, M., Strasser, A. W. M. & Hollenberg, C. P., Heterologous protein production in yeasts. Antonie van Leeuwenhoek (Int. J. Gen. Molec. Microbiol.), 62 (1992) 79-93. 22. Hadfield, C., Raina, K. K., Shashi-Menon, K. & Mount, R. C., The expression and performance of cloned genes in yeasts. Mycol. Res., 97 (1993) 897-944. 23. Weydemann, U., Keup, P., Gellissen, G. & Janowicz, Z. A., Ein industrielles HersteUungsverfahren von rekombinantem Hirudin in der methylotrophen Here Hansenula polymorpha. Bioscope, 3 (1995) 8-15. 24. Weydemann, U., Keup, P., Piontek, M., Strasser, A. W. M., Schweden, J., Gellissen, G. & Janowicz, Z. A., High-level secretion of hirudin by Hansenula polymorpha - - authentic processing of three different preprohirudins. Appl. Microbiol. Biotechnol., 44 (1995) 377-85. 25. Barnfield-Frej, A. K., Hjorth, R. & Hammarstr6m, A., Pilot scale recovery of recombinant annexin V from unclarified Escherichia c o l i homogenate using expanded bed adsorption. Biotechnol. Bioeng., 44 (1994) 922-9. 26. Chang, Y. K. & Chase, H. A., Expanded bed adsorption for the direct extraction of proteins. In Separations for Biotechnology 3, ed. D. L. Pyle. The Royal Society of Chemistry, London, 1994, pp. 106-12. 27. Chase, H. A., Purification of proteins by adsorption chromatography in expanded beds. TIBTECH, 12 (1994) 296-303. 28. Hansson, M., Stgihl, S., Hjorth, R., Uhl6n, M. & Moks, T., Single-step recovery of a secreted recombinant protein by expanded bed adsorption. Bio/Technology, 12 (1994) 285-8. 29. Th6mmes, J., Halfar, M., Lenz, S. & Kula, M.-R., Purification of monoclonal antibodies from whole hybridoma fermentation broth by fluidized bed adsorption. Biotechnol. Bioeng., 45 (1995) 203-11. 30. Spence, C., Schaffer, C. A., Kessler, S. & Bailon, P., Fluidized bed receptor affinity chromatography. Biomed. Chromatogr., 8 (1994) 236-41. 31. Bolivar, F., Rodriguez, R. L., Greene, P. J., Betlach, M. C., Heyneker, H. L., Boyer, H. W., Crosa, J. A. & Falkow, S., Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene, 2 (1977) 95-113. 32. Dohmen, R. J., Strasser, A. W. M., HOhner, C. B. & Hollenberg, C. P., An efficent transformation pro-
33. 34.
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cedure enabling long-term storage of competent cells of various yeast genera. Yeast, 7 (1991) 691-2. Gatignol, A., Durand, H. & Tiraby, G., Phleomycin resistance conferred by a drug-binding protein. FEBS Lett., 230 (1988) 171-5. Krstulovic, A. M. & Brown, P. R., Reversed-phase High-performance Liquid Chromatography- Theory, Practice and Biomedical Applications. Wiley & Sons, New York, 1982. Birnboim, H. C. & Doly, J., A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acid Res., 7 (1979) 1513-21. Sherman, F., Fink, G. R. & Hicks, J. B., Methods in Yeast Genetics. Cold Spring Harbor Press, New York, 1986. Maniatis, T., Fritsch, E. F. & Sambrock, J., Molecular Cloning. A Lab Manual. Cold Spring Harbor Press, New York, 1982. Laemmli, U., Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227 (1970) 680-5. Dairaku, K., Izomoto, E., Marikawa, H., Shioya, S. & Takamatsu, T., An advanced micro-computer coupled control system in a baker's yeast fed-batch culture using a tubing method. J. Ferment. Technol., 61 (1983) 189-96. Yano, T., Mori, H., Kobayashi, T. & Shimizu, S., Reusability of broth supernatant as medium. J. Ferment. Technol., 58 (1980) 259-66. Nishio, N., Tsuchiya, Y., Hayashi, M. & Nagai, S., A fed-batch culture of methanol-utilizing bacteria with pH Stat. J. Ferment. Technol., 55 (1977) 151-5. Jung, K. H., Park, M. H., Moon, H. M. & Rhee, J. S., Supplement of nutrients for effective cultivation of hepatitis B surface antigen-producing recombinant yeast. Biotechnol. Lett., 13 (1991) 857-62. Hinnen, A., Buxton, F., Chaudhuri, B., Heim, J., Hottiger, T., Meyhack, B. & Pohlig, G., Gene expression in recombinant yeast. In Gene Expression in Recombinant Microorganisms, ed A. Smith. Marcel Dekker, New York, pp. 121-93. Novick, P., Ferro, S. & Schekman, R., Order of events in the yeast secretory pathway. Cell, 25 (1981) 205-15. Schekman, R., Protein localization and membrane traffic in yeast. Ann. Rev. Cell Biol., 1 (1985) 115-43. Julius, D., Brake, A., Blair, L., Kunisawa, R. & Thorner, J., Isolation of the putative structural gene for the lysine-arginine endopeptidase required for processing of yeast prepro-ct-factor. Cell, 37 (1984) 1075-83. Gellissen, G., Janowicz, Z. A., Merckelbach, A., Piontek, M., Keup, P., Weydemann, U., Hollenberg, C. P. & Strasser, A. W. M., Heterologus gene expression in Hansenula polymorpha: efficient secretion of glucoamylase. Bio-Technology, 9 (1991) 291-5. Vlasuk, G. P., Bencen, G. H., Scarborough, R. M., Tsai, P. K., Whang, J. L., Maak, T., Camargo, M. J. F., Hirsher, S. W. & Abraham, J. A., Expression and secretion of biologically active human natriuretic peptide in Saccharomyces cerevisiae. J. Biol. Chem., 261 (1986) 4789-96. George-Nascimento, C., Gyenes, A., Halloran, S. M., Merryweather, J. P., Steimer, K. S., Masiarz, F. R. & Randolph, A., Characterization of recombinant human epidermal growth factor produced in yeast. Biochemistry, 27 (1988) 797-802. Gamier, J., Osguthospe, D. J. & Robson, B., Analysis of the accuracy and implications of simple methods
Aprotinin variants in Hansenula polymorpha for predicting the secondary structure of globular proteins. J. Mol. Biol., 120 (1978) 97-120. 5;1. Barthel, T. & Kula, M.-R., Rapid purification of DesPro(2)-Vall5-Leul7-aprotinin from the culture broth of recombinant Saccharomyces cerevisiae. Biotechnol. Bioeng., 42 (1993) 331-6. :~2. Barthel, T. & Kula, M.-R., Studies on the extraction of DesPro(2)-Vall5-Leul7-aprotinin from the culture broth of a recombinant Saccharomyces cerevisiae. Bioseparation, 3 (1993) 365-72. ~3. Tebbe, C. C., Vahjen, W., Munch, J. C., Feldmann, S. D., Ney, U., Sahm, H., Gellissen, G., Amore, R. &
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Hollenberg, C. P., Verbundprojekt ..Sicherheitsforschung Gentechnik - - Teil 1: Uberleben der Untersuchungsst/imme und Persistenz ihrer rekombinanten DNA. Bio-engineering, 6/94 (1994) 14-21. 54. Tebbe, C. C., Vahjen, W., Munch, J. C., Meier, B., Gellissen, G., Feldmann, S. D., Sahm, H., Amore, R., Hollenberg, C. P., Blum, S. & Wackernagel, W., Verbundprojekt Sicherheitsforschung Gentechnik - - Teil 2: Mesokosmenuntersuchungen und Einflul3 der Habitatbedingungen auf die Expression, l]berdauerung und Ubertragung des Aprotinin-Gens. Bio-engineering, 6/94 (1994) 22-6.
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Process Biochemistry, Vol. 31, No. 7, pp. 679-689, 1996 Copyright © 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0032-9592/96 $15.00 + 0.00
ELSEVIER
PI 1:S0032-9592
(96)00018-0
Production of Two Aprotinin Variants in
Hansenula polymorpha Christian Z u r e k , a E d w a r d Kubis, a P e t e r Keup, a Dietrich H6rlein, b Jiirgen Beunink, b J6rg T h 6 m m e s , c M a r i a - R e g i n a Kula, c Cornelis P. H o l l e n b e r g a & G e r d Gellissen a * "Rhein Biotech GmbH, Eichsfelder Strasse 11, 40595 Diisseldorf, Germany b Bayer AG, Pharma-Biotechnologie, 42096 Wuppertal, Germany c Institut for Enzymetechnologie, Heinrich-Heine-Universit~it Dtisseldorf, 52404 Jiilich, Germany a Institut for Mikrobiologie, Heinrich-Heine-Universit~it Diisseldorf, 40228 D~isseldorf, Germany (Received 9 January 1996; accepted 10 February 1996)
DNA-sequences coding for two DesPro(2) aprotinin variants were expressed in the methylotrophic yeast Hansenula polymorpha from a strong inducible promoter element derived from the MOX gene, a key gene of methanol metabolism. For secretion the coding sequences were fused to the KEX2 recognition site of the S. cerevisiae-derived MF~ 1 preproleader sequence. Correct processing of the precursor molecules and efficient secretion of the mature proteins was observed. A pH/pO2-controlled C-source feeding mode was applied for ferrnentations on a 10 litre scale. In cultures of a transformant strain harbouring 20 copies of the aprotinin expression cassette a yield of 350 mg/litre could be obtained. The secreted recombinant products were purified in a simple two-step isolation procedure employing an expanded bed adsorption as an initial purification step. Copyright © 1996 Elsevier Science Ltd
INTRODUCTION
tial as a therapeutic and diagnostic compound 6'7 forced the development of effective recombinant expression systems. Consequently, their establishment has been a major target of genetic engineering during the recent past after the respective gene sequences became available. A preferred production system is based on the yeast Saccharomyces cerevisiae 8-1° fusing the aprotinin sequence to a prepro-~-factor leader segment 11 for product secretion. High levels of incorrectly processed molecules have been encountered in a Pichia pastoris system due to steric features of the newly created fusion siteJ 2 Improvements have been described for S. cerevisiae-derived products deleting the proline residue in position 2 of the aprotinin chainJ °
The bovine pancreatic trypsin inhibitor aprotinin is a small protein of 6.5kDa and was initially isolated by Kunitz and Northrop. ~ The very basic (pI 10.5) single-stranded polypeptide consists of 58 amino acids cross-linked by three disulphide bridges. 2 It is found in a variety of bovine organs including liver and lung. 3 The natural aprotinin inhibits a range of proteases; its specificity can be modified by amino acid substitution within the reactive site. 4"5 The natural isolate has recently obtained FDA approval as a therapeutic. The high poten-
*To whom correspondence should be addressed. 679
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The methylotrophic yeast Hansenula polymorpha has been developed as an expression system for heterologous proteins. 13-~8 As a facultative methylotroph this yeast species is able to use methanol as the sole energy and carbon source; other possible carbon sources are glucose or glycerol. Upon addition of methanol into culture media or in media supplemented with glycerol in low concentrations, key enzymes of the methanol metabolism are strongly produced. The strong inducible promoter elements of the cloned key enzyme genes for the methanol oxidase (MOX) 19 and the formate dehydrogenase (FMD) 2° are used as components for heterologous gene expression providing a production process in recombinant strains which can be induced by compounds of a culture medium. After uptake, the heterologous DNA is stably integrated into the host's genome. Transformations result in a variety of strains harbouring a varying copy number of the integrated DNA in a head-to-tail arrangement. This stable multimeric integration of expression cassettes makes Hansenula polymorpha an ideal host for a genedosage dependent synthesis of foreign proteins. 16-19 As in other yeast expression systems a range of heterologous leader sequence from animal and yeast sources can be used for export targeting, among others the commonly used S. cerevisiae-derived MFo~I leader. 21-24 Purification of secreted proteins from whole fermentation broth demands a clarification step prior to further processing of the compound of interest. The solid-liquid separation step usually involves centrifugation or filtration followed by a concentration of the clarified liquid prior to subsequent purification procedures. Possible approaches to minimise product losses and time requirements of the early clarification and concentration steps are methods which integrate clarification and concentration into a single initial purification step. As such, fluidized bed adsorption has successfully been applied as a real chromatographic purification starting from unclarified broth. 25-3° In the present study we describe the engineering of recombinant H. polymorpha strains secreting two variants of aprotinin, namely the DesPro(2)-Lysl5 and the DesPro(2)-Argl5 aprotinin, and a rapid purification procedure of the secreted products from the culture broth of the engineered strains using
a fluidized bed adsorption as the initial purification step.
MATERIALS AND METHODS Strains and media
Plasmid constructs were propagated in E. coli strain HB10131 supplementing the media with chloramphenicol (30/~g/ml) when required for selection of transformants. The uracil-auxotroph mutant H. polymorpha strain RB11 deficient in oritidine-5'-phosphate decarboxylase (ura3) generated by the standard ethylmethane sulphate method served as standard hosts for the integration of the various plasmids constructed during the course of the present study. The strain was transformed as previously described. 32 In the case of supertransformation, transformation mixtures were plated on YNBplates containing phleomycin [700 ~,g phleomycin (Cayla) in 400/d], applied to the surface of hardened 20 ml agar platesY The antibiotic is applied after transformation allowing growth for about 4 h in advance. Positives were identified by culturing yeast clones under conditions of MOX-promoter derepression (see later) and determining the presence of aprotinin in the supernatants of the cultured cells by an ELISA using calibrated aprotinin samples for standardisation. A monoclonal antibody and aprotinin standards were kindly provided by Bayer AG Wuppertal. Test tubes (3 ml) and shake-flask (50 ml) cultures were grown in YNB [0.67% (w/v) Difco YNB] at 37°C supplemented with 1% (w/v) glycerol as carbon source for culture growth and at lower levels for heterologous gene expression (MOX-promoter derepression; see Introduction). When recombinant strains were grown at 101itre scale, a Biostat ER-10 fermenter (Braun, Melsungen) was charged with 7 litre of a synthetic medium as previously described, is supplemented with 0.2% (w/v) pepton 190, 0.3% (w/v) pepton 5 and 1.5% (w/v) glycerol. A 10% (v/v) Struktol J673 solution (Sichler, Germany) served for foam reduction. After steam sterilisation the medium was inoculated with 1 litre of an overnight culture of the recombinant strain grown in shake-fasks as described above, pH was adjusted with 30% phosphoric acid; base solution was 15% NH3 (w/v) and 50% glycerol (w/v). Glycerol was
Aprotinin variants in Hansenulapolymorpha
added as 50% (w/v) solution under pO2 control and via the base solution. During the consecutive last phase of fermentation a glycerol/methanol mixture (20:80) was fed in pulses for promoter induction. In the initial phase glycerol was added in pH-controlled pulses to maintain a concentration of 1-2% (w/ v). Then after 35 h upon consumption of glycerol a pOE-Controlled feed was initiated to maintain the glycerol concentrations between 0 and 0.2% (w/v) for the next 25 h. This was found to be optimal for promoter depression. During the ultimate induction phase C-source pulses provided glycerol concentrations between 0.0 and 0.2 and methanol concentrations between 0.2 and 1%. The culture conditions were maintained as follows: pH 4.5, 30°C, aeration rate 10 litre/min; stirrer speed 500 rpm. Fermentation on a 21itre scale was performed in a Biostat B fermenter (Braun, Melsungen, Germany) using the synthetic medium and conditions described above. Process parameters were controlled using the MFCS software (micro-multi control fermentersystem) supplied by the company. Glycerol and methanol concentration of fermenter samples were determined by gas chromatography (see below), aprotinin content by HPLC analysis34 and by the methods described before.
Isolation and analysis of nucleic acids Plasmid DNA was prepared from E. coli cultures by the alkaline extraction methodY Total yeast DNA was isolated from 50 ml cultures grown in YNB supplemented with 2% glucose according to Sherman et al. 36 DNA fragment separation and Southern analysis followed standard protocols. 37 Restriction enzymes, ligases and DNA-modifying enzymes were obtained from Boehringer Mannheim and employed according to the manufacturer's instructions. Gas chromatography Gas chromatographic quantification of glycerol and methanol was carried out on a Shimadzu GC-14A equipped with an integrator C-R4AX (Shimadzu), a SPBTM-5 column, length 15 m, thickness of layer 1-5 #m, ID 0.53 mm (Supelco) and a FI detector. Aliquots of centrifuged and sterile-filtered fermentation samples were diluted as 50 pl into 1 ml of 0.01% (v/v) 1-butanol. For separation of samples (sample volume
681
1 #1) the column was heated to an initial temperature of 100°C increasing to 250°C at a 15°C/min rate. Injector temperature: 320°C; detector temperature: 320°C. Helium served as carrier gas. A solution of 5 g/litre (w/v) glycerol and 1% (v/v) methanol were used as standards. HPLC analysis Centrifuged and sterile-filtered fermentation samples were diluted as 50 pl into 1 ml of a degassed buffer A [5% (v/v) CH3CN; 0.1% (v/v) TFA; 250 mM NaC104]. Aliquots (100 #l) of these samples were analysed on a Shimadzu CR4AX chromatopac equipped with a Controller 421A, a solvent delivery module 114M, a variable wavelength detector 165 adjusted to 214 nm, and an Altex 210 A valve (all from Beckman). Samples were separated on a 5 #m Spherisorb C8 column (20x4.6mm; PhaseSeparation) and eluted with a mixture of buffer A and a buffer B [80% (v/v) CH3CN; 0.09% (v/ v) trifluoroacetic acid (TFA), the percentage of buffer B increasing from an initial 10% to 35% at 2% min rate]. The flow rate was adjusted to 1 ml/min. Aprotinin content was determined by profile comparison with calibrated aprotinin standards.
Protein analysis Secreted proteins were separated on 15% SDS gels according to Laemmli38 and visualised by Coomassie staining. The N-terminus of aprotinin isolates was determined by automated liquid-phase sequence determination on a pulse liquid-phase peptide sequenator (Applied Biosystem 477A). Aprotinin purification For aprotinin purification 3200 ml of the culture broth (cell mass 100 g dry weight/litre) was diluted 1:1 with deionised water to 25 mS/cm conductivity and adjusted to pH 3.5. 300 ml (settled bed height 15 cm) of Streamline-SP cation exchanger (Pharmacia, Freiburg, Germany) were filled into a commercially available 5 cm diameter column for expanded bed adsorption (Streamline 50, Pharmacia, Freiburg, Germany). The adsorbent was fluidised at a linear flow rate of 5 cm/min and equilibrated with 10 column volumes of 20mM sodium citrate buffer pH 3.5 resulting in a three-fold bed expansion. Diluted broth (6400 ml) was applied at 5 cm/min, unbound proteins and resi-
682
C. Zurek et al.
dual biomass were subsequently washed from the expanded bed with equilibrating buffer. The bed was then allowed to settle and two elution procedures were performed in a fixed bed mode. In a first step employing 20 m u sodium citrate with 0"5 U sodium chloride contaminating proteins were desorbed. Crude aprotinin was eluted using 20 m u sodium citrate with 0.9 M sodium chloride. The matrix was regenerated with 2 M sodium hydroxide in a fixed bed mode as well. The aprotinin-containing eluate was supplemented with 0.1% (v/v) of TFA and applied to a RP 18 Lichroprep HPLC column (Merck, Darmstadt) at 2 cm/min without further pre-treatment. Pure aprotinin was eluted using a gradient from 0.1% TFA in aqueous solution to 50% of 0.1% (v/v) TFA in 40% (v/v) isopropanol. The aprotinin-containing fractions were desalted by gel filtration on a Sephadex G25 column (Pharmacia, Freiburg, Germany) and freeze-dried.
medium to obtain mitotically stable transformants bearing the integrated foreign DNA. In the case of phleomycin at a concentration of 35#g/ml. Out of several hundred clones
BamHI
i ltindlII /EcoRl
Pstl
(A)
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n,RM,y A.~. MOX-P"~
ttindIII ~
Psi'
cam/~/ EeoRI PstIi
RESULTS
(B) Plasmid construction A 0.5 kb DNA fragment containing a synthesised MF~l-leader/aprotinin fusion (desPro(2)Argl5) was cloned into the multiple cloning site of plasmid pRBMOX located between a MOXpromoter and a MOX-terminator element thus constituting an expression cassette for the secretion of aprotinin. The construction results in the plasmid pRBMOXApro (see Fig. 1A). For construction of the plasmid pRBMAproPhle a phleomycin resistance gene was inserted into this vector (Fig. 1B). For generation of the respective plasmid for the expression of DesPro(2)-Lysl5 the fusion fragment was subcloned into a M13 vector and mutagenised by site directed mutagenesis. The successful mutagenesis was controlled by sequencing, and the newly obtained fragments were inserted into the basic expression vectors as described before. Generation of aprotinin-secreting
H. polymorpha strains After rounds of transformation with the two pRBMOXApro constructs described before (for details see the Materials and Methods section), colonies secreting aprotinin were isolated and sequentially grown for 70 generations in selective (YNB) and for two passages in rich (YPD)
;BamHI SalI
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/.
),.co.,
IlindIII(I IIAIISIpRBMAproPhle =o.-,ll:"" 1
io,,o
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.......
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Fig. 1. Physical maps of the expression vectors for aprotinin used in this study. The various MFctl/aprotinin fusions were inserted as EcoRI/Bg/II fragments into the multiple cloning site of the H. polymorpha expression/ integration vector pRBMOX separating a MOX-promotor (MOX-P.) and a MOX-terminator (MOX-T.) sequence. The plasmid contains the following additional components: o r / ( O R I ) and a chloramphenicol resistance (cam) gene for selection and propagation in E. coli and a Hansenula autonomously replicating sequence (HARS1) and a URA3 gene (URA3) for propagation and selection in the uracil-auxotrophic H. polymorpha host strain RBll. The resulting plasmid is pRBMOXApro (A). For retransformarion a phleomycin resistance gene obtained from the plasmid pUT332 (CAYLA) was inserted to result in the plasmid pRBMAproPhle (B).
Aprotinin variants in Hansenula polymorpha
obtained examples representative for the different constructs were selected for a more detailed analysis. In preliminary expression studies in fermentations at a 50 ml scale clones were grown in 2% glycerol, secretion of aprotinin was monitored after 65 h ranging from 5 to 20 mg/litre. Examination of genomic DNA by Southern hybridisation identified several strains with different copy numbers of the integrated expression cassette. To estimate the copy number, DNA was restricted with SalI/XbaI, transferred to nitrocellulose filter, and hybridised to a 32p-labelled MOX-promoter probe. The signal intensity of calibrated DNA dilutions could be compared to that of the intrinsic single copy control derived from the host MOX gene and provided an estimation of heterologous gene content. The copy number was determined as a range from 1 to 16 copies in the various strains analysed. In case of a DesPro(2)-Argl5 strain a particular isolate bearing 16 copies of the heterologous expression cassette was retransformed with plasmid pRBMAproPhle and transformants were selected according to the newly introduced resistance. In the example documented in Fig. 2 the selected strain 23-3 is analysed and identified as having acquired two additional copies of the aprotinin expression cassette. In the case of DesPro(2)-Lysl5 variant transformants harbouring a gene dosage of between one and 14 copies could be identified (see Table 1). The integrated DNA was found to be mitotically stable after passaging for more than 80 generations (data not shown). For further studies we focused on the analysis of a single transformant of each construct. The selected strains habour 18 copies (supertransformant of the DesPro(2)-Argl5 type) and 14 copies (DesPro(2)-Lysl5-type), respectively (Fig. 2 and Table 1). The supertransformant expressing the DesPro(2)-Argl5 variant is now designated as strain I, the transformant for the expression of the alternative DesPro(2)-Lysl5 aprotinin as strain II. Expression and properties of aprotinin secreted from strains I and II In a comparative analysis the two strains were cultivated at a 2 litre scale as described in the Materials and Methods section. After 26h under MOX-promoter derepression culture supernatants were harvested and analysed for
683
a
b
O Fig. 2. Copy number determination of the heterologous DNA in recombinant strains. Genomic DNA from a selected retransformed strain (strain I) was digested with SalI/Xba I. Standardised dilutions of the various restriction samples were separated through 0.8% agarose gels, transferred to nitrocellulose filters and hybridized to a 32p-labelled MOX-promotor fragment, comparing the intensity of the resulting hybridisation signals derived from the heterologous fusions with that of the single copy MOX gene. The strain was estimated to contain 16 copies derived from the first transformation with pRBMOXApro and two further copies from the retransformation with pRBMAproPhle. The faint upper signal corresponds to the intrinsic single copy MOX gene; the strongest signal in the middle position to the heterologous fusion resulting from the transformation with pRBMOXApro and that in the lower position to that from retransformation with pRBMAproPhle. (a) Size marker (EcoRI/HindlII digest of 2 DNA). (b) Sal/I/Xba I digest of genomic DNA from strain I (undiluted, dilutions 1:2; 1:4; 1:8; 1:16 and 1:32; from left to right).
Table 1. Properties of selected aprotinin-secreting strains
Strain
Aprotinin variant
Copy number
Productivity Correctly (mg/litre) processed product
(%)
Strain I Strain II
DesPro(2)Argl5 DesPro(2)Lysl5
16 + 2
350
82
14
270
80
C. Zurek et al.
684
aprotinin content. The highest accumulation of aprotinin was observed in the culture of strain I and determined as 150 mg/litre, whereas strain II produced 100 mg/litre, respectively, roughly reflecting the difference in aprotinin gene dosage. Nevertheless, all constructs provide for an efficient secretion confirming that both MF~I/ aprotinin preprostructures are recognised by the secretory system of the methylotrophic host. A comparative HPLC-analysis revealed that the secreted aprotinin consists of a mixture of different species. In all cases the correctly processed full length form represents the majority product in the medium ranging from 80 (strain II) to 82% (strain I) of the overall aprotinin yield (Table 1). Determination of N-terminal amino acid sequences revealed different N-terminal extensions with a main peak harbouring aprotinin of a correctly processed N-terminus (data not shown; see also analysis of purified aprotinin from a 10 litre fermentation).
of the substrate, glycerol was kept at a low level with a pO2-controlled feed to promote heterologous gene expression (condition of MOX-promoter derepression). After a total fermentation time of 50-70h a feed of six consecutive pulsed additions of a methanol/ glycerol mixture (80% methanol, 20% glycerol) for MOX-promoter induction was initiated. Details of the fermentation conditions are given in the Materials and Methods section and in the legend of Fig. 3. After a total fermentation time of 80-100 h, the aprotinin content of the supernatant was analysed as 320mg/litre. The productivity of strain II could be optimised to 270mg/litre applying a similar fermentation regimen (Table 1). Purification of aprotinin accumulated in the culture medium
The purification of aprotinin in a simple twostep procedure was made possible by the fact that only very low levels of contaminating secretory proteins from the H. polymorpha host are present in the culture broth. From the beginning a high product titre simplified the chromatographic purification. Application of the whole culture broth to the cell matrix necessitated a 1:1 dilution of the broth. In this way the conductivity of the medium was lowered to a value where adsorption of aprotinin to the ion
Fermentation studies at 10 litre scale
Productivity of the two strains was further analysed in fermentation runs at 10 litre scale. In the analysis documented in Fig. 3 strain I was cultured following a two-carbon source mode. The cells were first grown in 1-2% glycerol to a dry weight of 30 g/litre in a pHcontrolled feed mode. Then upon consumption
350
~l~.growth
phase pH-stat
.~ r
derepression phase ~
~
induction phase
500
time control
pO2-stat
300
400
. ,X . . . . X- - -X . . . . . . . . . . o 250
~
,x"
200
~150
• . X" "
..___~'"~
1
"
"
-
300
x"
200
O
.-
~
P
100 100
50 0
I0
20
30
40
50
60
70
80
90
Fermentation Ume [h]
I---x--- Celldensity
A Aprotinin I
Fig. 3. Fermentation of strain 1 at a 10 litre scale. A fermentation run was carried out as described in the Materials & Methods section. The fermentation started with 2% glycerol at the beginning and a pH-stat-glycerol feeding mode during the initial growth phase of the culture. After 35 h and the consumption of the carbon source a pO2-controlled feed was initiated resulting in glycerol concentration between 0-0 and 0"2% (w/v) (derepression of the MOX-promoter). After 60 h a methanol/glycerol mixture was fed in six consecutive pulses. Timed fermentation samples were taken and analysed for aprotinin content in the supernatant and other fermentation parameters. After a fermentation time of 85 h the culture broth was harvested and aprotinin was purified from the supernatant.
Aprotinin variants in Hansenula polymorpha
exchanger was favoured; furthermore the viscosity of the broth was reduced, such that the bed did not expand beyond the upper end of the column when being fluidized with the bio~nass-containing sample. The diluted culture broth had a biomass content of 5% dry weight thus being within the range recommended. 25 During adsorption total aprotinin was captured ,)n the ion exchanger. The two-step elution ~rotocol produced 3.8-fold purified and sevenfold concentrated aprotinin at a 68% yield. The clarification of the cell-containing broth was ~atisfactorily achieved in addition to the chromatographic success. Therefore, after addition of 0.1% TFA the eluate was applied to the HPLC column without further treatment. The ~eparation of incorrectly processed aprotinin ~vas accomplished in the RP-HPLC step. HPLC, SDS-PAGE and N-terminal sequencing confirmed the fidelity and homogeneity of the recovered isolate (Figs 4 and 5). The results of the purification are summarised in Table 2.
DISCUSSION
Fed-batch fermentations of particular H. polymorpha strains described in this report followed a two-carbon source mode adding suitable amounts of glycerol and methanol to the broth. Feeding of these two compounds followed a regimen which provides an optimal growth during the initial growth phase and which achieves high product yields during a two-phase production by first derepressing and then inducing the MOX-promoter used for heterologous gene expression. During the initial growth phase a high glycerol content of 1-2% was maintained by a feeding mode coupled to base consumption. In the following derepression phase the glycerol was added by a p O z - c o n trolled feeding mode. Similar strategies have been described using pO2, pH or product concentrations for 'indirect' feed-back c o n t r o l . 39-42 This results in a simple process which is relatively unsusceptable to potential interferences. The methylotrophic yeast H. polymorpha exhibits favourable properties for heterologous gene expression. One major advantage is the potential of this microorganism to secrete large amounts of protein products into the medium. 15-~8 As unicellular eukaryotic organisms, yeasts
685
share to a large extent the basic characteristics of the secretory pathway ubiquitously found in organisms ranging from fungi to mammalians. As a consequence, a primarly translation product can correctly be processes irrespective of the source of a leader sequence used for secretion. 21-24"43 Such a leader may comprise a short 20 amino acid sequence targeting the polypeptide to the endoplasmatic reticulum (ER) and which is cleaved off upon entry into this compartment by a signal peptidase. 44"45 Alternatively, it may consist of a preprosegment. For processing of the latter further proteolytic cleavage of the imported pro-polypeptide is required by a dibasic protease of the Golgi apparatus equivalent to the S. cerevisiae endopeptidase F (gene product of the KEX2 gene).4('
A214
A
2-
1-
lb
ttmi~
1'o
t (rain)
1"
g
Fig. 4. HPLC-analysis of aprotinin samples derived from the fermentation described in Fig. 3. HPLC conditions are as described in the Materials and Methods section. (A) Supernatant of the culture broth. The two arrows indicate fractions containing incorrectly processed or degraded aprotionin molecules. (B) Purified aprotinin obtained after the final purification step.
C. Zurek et al.
686
1
2
3
4
5
i I
,q
Fig. 5. SDS-PAGE analysis of aprotinin samples. Denatured protein samples were separated through 15% SDS-PAGE gels and visualised by silver staining. Lane l, Pharmacia low molecular weight marker corresponding o 17.2, 14.6, 8.2, 6.4 and 2.5KDa, respectively; lane 2, supernatant from the fermentations of strain I (see Fig. 3); lane 3, crude aprotinin after the first purification step (0"9 M NaCI eluate from the Streamline SP column); lanes 4 and 5 purified aprotinin samples after the first and the second reversed phase chromatography, respectively.
A variety of leader sequences has been employed in yeast expression systems inclusive of H. polymorpha following the general design of the two basic types, many of them originating from a heterologous source. 21-25"47 However, not all heterologous leader sequences function in yeasts. For instance, those of leech echistatin and human tissue plasminogen activator do not direct secretion in S. cerevisiae.48"49 Furthermore, correct processing might be hampered by structural or steric features of the newly created fusion. Examples for such heterogeneity are available for both basic leader types, among others for aprotinin produced in the methylotrophic yeast P. pastoris. 12 Correct processing can be predicted to some extent by empirical rules. 5° This prediction suggested the deletion of the proline residue in position 2 to create a processing site more suited for KEX2 processing. In the study presented here we employed the Table 2. Summary of the aprotinin purification protocol
Sample
Harvest Streamline SP eluate (0-9 M NaC1) RP 18 HPLC Sephadex G25
Aprotinin concentration (mg/litre )
Purification (fold)
YieM (%)
202 1412
1 3.8
100 76
719 543
5.45 5.5
36 35
preprosegment of MF~I which seemed more suitable to us for direct secretion of smaller products like aprotinin from a H. polymorpha host and which has successfully been applied for the secretion of hirudin from this microorganism 23'24 and for export of aprotinin from S. cerevisiae. Closer inspection and comparison of the secreted products revealed that expressions of both preproaprotinin fusion result in a high share of correctly processed mature protein, contrasting with the situation in P pastor& where an aprotinin sequence including the proline was expressed. Aprotinin species with N-terminal-amino acid extensions make up 15% of the overall aprotinin yield demonstrating that the N-terminal modification has led to a considerable improvement. Secretion provides an attractive mechanism for product recovery without having to break open cells, especially since the methylotrophic host secretes genuine polypeptides in low abundance only. Accordingly, a relatively simple combination of chromatographic procedures can be employed for the purification of secreted aprotinin mainly to exclude incorrectly processed molecules from the desired isolate. The classical strategy used for the purification of recombinant aprotinin is based on an initial clarification of the growth medium, a cation exchange chromatography or affinity chromatography as first enrichment and a final purification by reversed phase HPLC. After desalting by gel filtration and freeze-drying, a pure and correctly processed product is obtained. 5j'52 To simplify the purification process the first three steps were combined in the present study by an expanded bed adsorption employing a commercially available cation exchange material suitable for expanded bed adsorption (Streamline SP). The resulting eluate was found to be ready for direct application to the subsequent purification. The purified aprotinin appeared to be homogeneous and correctly processed. The engineered H. polymorpha strains further attests to the potential of this system for a commercially competitive production processes. The relatively poor productivity of the two strains used in this study is probably the result of the expression cassettes being present in low copy numbers. In other recent examples H. polymorpha strains have been obtained bearing 40 copies of a heterogeneous gene or more which
Aprotinin variants in Hansenula polymorpha
secrete a recombinant product in the gram range. Since the production capabilities for a given protein is obviously correlated to some extent to the dosage of the respective gene in the expressing host it appears likely that further high-copy transformants can be selected for both strain types exhibiting higher productivities. On the other hand, the produced aprotinin might impose a physiological stress on the yeast restricting transformants to a gene dosage and a productivity as observed in the described examples. H. polymorpha provides several advantages as production organism for a compound considered for administration to humans. The mitotically stable introduction of foreign genes creates strains suited for a reproducible fermentation process under non-selective conditions. In risk assessment studies the ecological safety of aprotinin-secreting strains has been s h o w n . 53'54 The characteristics and the performance of the aprotinin-producing strains described in this publication attest together with previous examples TM that the H. polymorpha expression system meets the general demands and prerequisites for industrial application where a system of choice should produce an optimal amount of authentic bioactive material in a reproducible and stable production process. ACKNOWLEDGEMENTS
The authors gratefully acknowledge the financial and logistic support by the Bayer AG, Wuppertal. We thank Mr K.-P. M~ider (Bayer AG) and J. D0rkop (Rhein Biotech GmbH) for their excellent technical assistance and D. Kinzelt, lET, Heinrich-Heine Universit~it Diisseldorf for determination of amino acid sequences. REFERENCES 1. Kunitz, M. & Northrop, J. H., Isolation from beef pancreas of crystalline trypsinogen, trypsin inhibitor, and an inhibitor trypsin compound. J. Gen. Physiol., 19 (1936) 991-1007. 2. Anderer, F. A. & HOrnle, S., The disulphide linkages in kallikrein inactivator of bovine lung. J. Biol. Chem., 241 (1966) 1568-72. 3. Fritz, H. & Wunderer, G., Biochemistry and applications of aprotinin, the kallikrein inhibitor from bovine organs. Arzneim. Forsch./Drug Res., 33 (1983) 479-94. 4. Dietl, T., Huber, C., Geiger, R., Iwanaga, S. & Fritz,
687
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Hollenberg, C. P., Verbundprojekt ..Sicherheitsforschung Gentechnik - - Teil 1: Uberleben der Untersuchungsst/imme und Persistenz ihrer rekombinanten DNA. Bio-engineering, 6/94 (1994) 14-21. 54. Tebbe, C. C., Vahjen, W., Munch, J. C., Meier, B., Gellissen, G., Feldmann, S. D., Sahm, H., Amore, R., Hollenberg, C. P., Blum, S. & Wackernagel, W., Verbundprojekt Sicherheitsforschung Gentechnik - - Teil 2: Mesokosmenuntersuchungen und Einflul3 der Habitatbedingungen auf die Expression, l]berdauerung und Ubertragung des Aprotinin-Gens. Bio-engineering, 6/94 (1994) 22-6.