0 1989 by The American Society for Biochemistry and Molecular Biology, Inc. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 264, No. 8, Issue of March 15, pp, 4367-4373, 1989 Printed in U.S.A.

Expression of Human Parathyroid Hormone in Escherichia coli* (Received for publication, October 12, 1988)

Edgar Wingender, Gisela BerczS, Helmut Blocker, Ronald Frank, and Hubert Mayer From the Gesellschaft fur Biotechnologische Forschung, 0-3300 Braunschweig, Federal Republic of Germany

Human parathyroid hormone (PTH) has been ex- pressed in Escherichia coli as a cro-8-galactosidase- hPTH fusion protein under temperature-sensitive con- trol of the X phage PR promoter. The lacZ gene has been truncated to a different extent revealing an optimal length of the prokaryotic peptide portion between 199 and 407 amino acid residues. Up to 250 mg of pure fusion protein have been obtained from 1-liter E. coli culture by stepwise solubilization with urea. The link- age between the prokaryotic and the eukaryotic pro- tein moiety consists of an Asp-Pro peptide bond and therefore is easily cleavable by acid treatment. A sim- ple procedure for the purification of the hormone is described. The resulting recombinant hormone reacts with anti-PTH antibodies and stimulates renal adenyl- ate cyclase identically to bovine or human PTH.

Parathyroid hormone (PTH)’ is an 84-amino acid residue peptide and one of the main regulators in maintenance of calcium homeostasis. It acts primarily on kidney and bone cells stimulating calcium back resorption or calcium mobili- zation, respectively. Moreover, it also leads to an enhance- ment of bone remodeling processes (for review see Potts et al., 1982). This has been demonstrated by its ability to stim- ulate cell proliferation of chondrocytes or osteoblasts in ap- propriate i n vitro cell systems (van der Plaas et al., 1985; Lewinson and Silbermann, 1986; Kawashima et al., 1980; Burch and Lebovitz, 1983). Thus, parathyroid hormone ex- hibits differential catabolic as well as anabolic effects. Some recently published studies revealed the promising effect of therapeutical application of PTH as an agent against osteo- porosis (Slovik et al., 1987). For this purpose an expression system would be of interest providing sufficient material for further studies and, possibly, clinical applications. Further- more, such an expression system would provide a basis for protein engineering attempts to investigate structure-function relationships for this multifunctional peptide hormone as well as for designing hormone variants for special tasks, e.g. to promote either the calcium mobilizing or the bone (re-)con- structing effect.

Previous attempts in our laboratory to achieve direct

* This work was part of Protein Design Project 0387069 supported by the Bundeministerium f i r Forschung und Technologie of the Government of the Federal Republic of Germany. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by the Deutsche Forscbungsgemeinschaft. ’ The abbreviations used are: PTH, parathyroid hormone; (h)PTH,

(human) parathyroid hormone; Gpp(NH)p, &y-imidoguanosine 5’- triphosphate; Nle, norleucine; SDS-PAGE, sodium dodecyl sulfate- polyacrylamide gel electrophoresis; HPLC, high pressure liquid chro- matography.

expression of human PTH in Escherichia coli yielded only very limited amounts (up to 500 wg/liter) due to RNA and protein instability (Breyel et al., 1984; Morelle and Mayer, 1988). Another recently published direct expression system similarly yielded a maximal production of 200 rglliter culture medium. However, the product was shown to be NHa-termi- nally heterogeneous and required a relatively intricate puri- fication procedure leaving maximally 10 pg/liter culture (Rab- bani et al., 1988).

To circumvent these problems apparently inherent to direct PTH expression in E. coli, we constructed a series of expres- sion plasmids allowing synthesis of a cro-@-galactosidase- hPTH fusion protein. They contain a variable prokaryotic portion and a fusion site which is amenable to chemical cleavage of the resulting hybrid protein by acid hydrolysis. Subsequently, we purified the hormone and assayed for its biological activity.

EXPERIMENTAL PROCEDURES

heim, West Germany) or from GIBCO and Bethesda Research Lab- Materials-Restriction enzymes were from Boehringer (Mann-

oratories. Anti-PTH antibodies were a kind gift from Prof. Hesch, Medizinische Hochschule Hannover. Horseradish peroxidase-conju- gated detection antibodies (rash anti-sheep and goat anti-rabbit) were purchased from NORDIC (Tilburg, The Netherlands). Bovine and human PTH(1-84) as well as [Nle’, Nle”, Tyr3‘]bPTH(1-34)amide were obtained from Sigma. All other chemicals were p.a. grade and obtained from Merck (Darmstadt, Federal Republic of Germany, (F. R. G.)). The bacterial strain used for the expression studies is E. coli N4830 which carries the X prophage cI857 (Gottesman et al., 1980).

Vector Construction-Starting from a genomic cosmid clone of the human PTH gene (Mayer et al., 1983, 1984) we isolated the 260 base pair NsiI-XbaI fragment encoding the amino acid residues 10-84. Its 3’-end was ligated to the XbaI site of the pEX vector. The DNA double strand coding for the NHz-terminal 9 amino acids plus one extra proline codon was chemically synthesized with cellulose discs as segmental supports as is described elsewhere (Frank et al., 1983, 1987). It was cloned between the NsiI site of the hPTH gene and the EcoRV site of pEX. The resulting plasmid transformed into E. coli N4830. All cloning procedures were performed according to the stand- ard protocols of Maniatis (1982).

Expression, Extraction, and Cleavage of Fusion Protein-1 liter of LB medium containing 50 Fg/ml ampicillin was inoculated with 10 ml of an overnight culture of the E. coli strain harboring the expres- sion plasmid. If not stated otherwise, this culture was shaken at 30 “C for 4 h; at this time an optical density of approximately 0.5 (550 nm) was reached. Subsequently, expression was induced by shifting the temperature for 16 h to 42 “C. During this period, the OD,,, increased further up to 4. The bacteria were harvested by centrifugation (15 min, 6000 rpm, Beckman JA-10 rotor) and resuspended in 75 ml of 40 mM Tris/HCl, pH 8.0, 5 mM EDTA, 0.3 mg/ml lysozyme. After 1.5 h at 0 “C, 75 ml of 20 mM Tris/HCl, pH 7.4, 20 mM MgClz were added, the mixture was adjusted to 33 pg/ml DNase I (grade 11, Boehringer-Mannheim (F. R. G.)) and left on ice for 1.5 h. Subse- quently, 15 ml of 10% sodium deoxycholate were added and after 30 min on ice the mixture was centrifuged (20 min, 8500 rpm in a Beckman JA-14 rotor). The pellet was resuspended in 10 ml of 2 M urea, 20 mM Tris/HCl, pH 7.4, and left on ice for 1 h. Centrifugation (20 min, 8500 rpm) yielded another pellet which was resuspended in 10 ml of 9 M urea, 20 mM Tris/HCl, pH 7.4, 2 mM dithiothreitol.

4367

4368 Recombinant Human Parathyroid Hormone After 1 h at room temperature, this suspension was centrifuged (30 min, 9000 rpm). The last step was repeated until virtually all extract- able protein was resuspended.

For cleavage of the fusion protein, the 9 M urea extract was mixed with an equal volume of concentrated formic acid and incubated at 37 "C for 3-5 days. It was subsequently dialyzed at 4 "C against 5 liters of water containing an appropriate amount of NaOH to neu- tralize the formic acid. When the pH of the dialysis bath changed to neutral, dialysis proceeded against 5 liters of water (with two changes) and 10 mM ammonium acetate.

Purification of the Recombinant hPTH-The solution was read- justed to 8 M urea and loaded onto a column of carboxymethyl- cellulose (CM52, Whatman) at 1 mg of protein/ml resin; the ion exchanger had previously been equilibrated with 8 M urea, 10 mM ammonium acetate. After careful washing with the same buffer, the hormone was desorbed with 8 M urea, 100 mM ammonium acetate. The PTH-containing eluate was subsequently dialyzed against 10 mM acetic acid and concentrated either by lyophilization or by am- monium sulfate precipitation (3 M (NH&S04) followed by dialysis against 10 mM acetic acid to remove residual urea or salt. For

additional purification by reversed-phase HPLC, this material was loaded onto a 4.6 X 25 cm C4 column from Vydac (Heseteria, CA) connected to HPLC equipment from LKB (Bromma, Sweden). Elu- tion was performed with an acetonitril gradient in 0.1% trifluoroacetic acid. Resulting fractions were concentrated with a Speed-Vac and analyzed by SDS-PAGE for contaminating proteins.

Analytical Procedures-Protein concentrations were measured ac- cording to Bradford (1976) using the Bio-Rad assay with bovine serum albumin as standard. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was as described by Laemmli (1970) on 17% acrylamide, 0.34% bisacrylamide gels.

Immunoblotting was essentially as described (Towbin et al., 1979). Binding of anti-PTH antibodies proceeded overnight a t room tem- perature, binding of peroxidase-conjugated antibodies (rash anti- sheep and goat anti-rabbit) for 1 h each. Staining was with I-chloro- 1-naphthol as substrate.

After hydrolysis of 27 pg (28 nmol) recombinant hPTH with 6 N HCl for 24 h a t 110 'C in either the absence or presence of 7% thioglycolic acid, analysis of the amino acid composition was per- formed with an amino acid analyzer LC5001 (Biotronik) connected

3.0

\ hPTH-'

"_ - B-Gal -1 ... Mat Val Gln Alp Pro

1 . 5

- hPTH +

I

Ser Val Ser Glu 1 1 0 Gln Leu Met Hi8 A8n ... . . . ATG GTG CAG GAT CCG TCT GTG AGT GAA ATT CAG CTT ATG CAT AAC . . . . . . TAC CAC GTC CTA GGC AGA CAC TCA CTT TAA GTC GAA TAC GTA TTG . . .

Barn H I Nul 1

FIG. 1. Expression plasmids for hPTH. In pEX-PPTH, the gene for the human PTH (cross-hatched) which was elongated at i ts 5' end by a proline codon (CCG) was fused to the truncated cro-lacZ gene which encodes the hybrid protein cro-P-galactosidase(1-407) (hatched). Transcription is under the control of the PR promoter (black) in the indicated direction. Starting with this plasmid, we deleted the HpaI-HpaI, the HpaI-BamHI, or the HindIII- BamHI fragment to obtain the plasmids pEW1, pEW2, or pEW3, respectively. At the bottom, the sequence of the P-galactosidase-hPTH junction is given. The DNA sequence shown in italics was chemically synthesized.

Recombinant Human Parathyroid Hormone 4369

with a Shimadzu C-R3A integrator. Determination of the amino acid sequence was achieved using a 470A protein sequencer which is connected on-line with a 120A PTH analyzer (Applied Biosystems).

Adeny!ate Cyclase Assay-Porcine renal cortical membranes were prepared and enriched by differential centrifugation and ultracentrif- ugation on continuous Percoll density gradients by the method of Mohr and Hesch (1980).

Adenylate cyclase activity was measured by the formation of CAMP from ATP according to Mohr and Hesch (1980) with some modifi- cations. Incubation was carried out at 30 “C for 30 min in a mixture consisting of 50 mM Tris/HCl, pH 7.4, 1.8 mM ATP, 8 mM creatine phosphate, 100 pg of creatine phosphokinase, 1 mM 3-isobutyl-l- methylxanthine, 4.5 mM MgC12,15 mM KCI, 10 p~ Gpp(NH)p, 50 pg of membrane protein, and variable concentration of PTH in a total volume of 200 pl. The reaction was initiated by the addition of the membrane protein and terminated by heating the samples to 85 “C for 10 min. The samples were centrifuged for 5 min a t 9000 X g and the CAMP content in the supernatants was measured in a [‘HICAMP radioassay (Amersham, Braunschweig, F. R. G.).

RESULTS

Vector Construction-Starting from one of the pEX expres- sion vectors described by Stanley and Luzio (1984), we in- serted the gene for the human PTH between the restriction sites for EcoRV (immediately 3‘ to the Asp(407) codon of the cro-&galactosidase fusion protein) and XbaI (Fig. 1) as it is known from similar vector systems that this truncation is not disadvantageous for expression (Broker, 1986). The PTH coding region was obtained from a human genomic clone (Mayer et al., 1984), taking the sequence between the internal NsiI site within the codon for His-9 and the XbaI site in the 3“flanking region (Vasicek et al., 1983). The DNA sequence encoding the correct NHa-terminal 9 amino acid residues up to the NsiI site was chemically synthesized as a DNA duplex using a previously published method (Frank et al., 1983,1987):

5”CCG TGT GTG AGT GAA ATT CAG CTT ATG CA -3’ 3”GGC ACA CAC TCA CTT TAA GTC GAA T -5 ‘

Deviating from the original human base sequence, we substi- tuted in this part of the gene the isoleucine 5 codon ATA, which in E. coli is very rarely used (Maruyama et al., 1986), by the more frequent ATT. Furthermore, the synthetic DNA duplex was supplemented a t its 5’ end with an additional proline codon (Fig. 1). The resulting plasmid (pEX-PPTH) codes for a fusion protein which contains an acid-labile Asp- Pro linkage between the prokaryotic and the eukaryotic moiety. Thus, acid hydrolysis should release a human para- thyroid hormone with an extra proline residue at its amino terminus (subsequently referred to as Pro-’-hPTH(1-84) or simply as “recombinant PTH”).

This construct which contains a new BamHI site at the 8- galactosidase-hPTH gene junction was subsequently short- ened by the following in frame deletions: (i) deletion of the HpaI-HpaI fragment removed the amino acid residues 179- 386 from the cro-P-galactosidase portion (pEW1); (ii) restric- tion with HpaI and BamHI, fill-in of the latter site and religation, deleted amino acid residues 179-406 (pEW2); (iii) cleavage with Hind111 and BamHI and religation of the large fragment after fill-in reaction removed the codons for residues

Expression-All the constructs mentioned were trans- formed into E. coli strain N4830 harboring a temperature- sensitive X repressor in its genome (cI857) (Gottesman et al., 1980). Therefore, growth at 30 “C completely represses the X PR promoter but allows induction of the PR-controlled gene by temperature shift to 42 “C.

Total protein extracts of small-scale cultures of pEX- PPTH, pEW1, and pEW2 displayed very clear induction- specific bands when analyzed on SDS-polyacrylamide gel

11-406 (pEW3).

electrophoresis (Fig. 2). When compared to pEX-PPTH, the lower yield of fusion protein obtained from PEW1 was nearly compensated by the increased portion of PTH sequence (17 and 30%, respectively; see below for quantification). A real reduction in expression of fusion protein as well as of net PTH sequence was observed for pEW2 (Fig. 2), and pEW3 exhibited no detectable levels of the “mini-fusion” protein indicating a critical minimum length of the prokaryotic por- tion (not shown).

Frequently, overexpressed p-galactosidase fusion proteins are deposited in E. coli cells as inclusion bodies (for review see Marston, 1986) and this has also been observed by electron microscopy for the cro-p-galactosidase-PTH hybrid protein described here (not shown). We therefore attempted to ini- tially purify the inclusion bodies by fractionated extraction with 0, 2, and 9 M urea after cell lysis and DNA digestion with DNase I. The fusion protein was exclusively obtained in the 9 M urea extract (Fig. 3, lane 6) . Due to its high purity the protein content of this fraction was used as criterion for the expression yield in subsequent optimization experiments.

The procedure described above also worked well for the preparation of fusion proteins which were synthesized in bacteria harboring either plasmid PEW1 or pEW2. In the latter case, however, the urea concentration of the second wash step had to be lowered to 1 M urea since the shorter fusion protein is more easily solubilized by this denaturant.

1 2 3 4 M - 64

- 45

V - 29

- 22

- 12.5

- 6 . 5

kDa FIG. 2. Expression of cro-&galactosidase fusion proteins. 2-

ml cultures of E. coli harboring the expression plasmid shown in Fig. 1 were grown for 1 h a t 30 ‘C and 4 h a t 42 ‘C. The cells were harvested, the proteins solubilized with 1% SDS and analyzed by electrophoresis on a 17% SDS-polyacrylamide gel. The positions of the induction-dependent fusion proteins encoded by pEX-PPTH ( l a n e 2), PEW1 (lane 3), and pEW2 (lane 4 ) are indicated by the arrows on the left. The protein pattern of noninduced control cells (5 h a t 30 “C) is shown as well ( l a n e 1 ). Marker proteins ( l a n e M) are bovine serum albumin, ovalbumin, carboanhydrase, trypsin inhibitor from soybean, cytochrome c, and trypsin inhibitor from lung. The molecular masses are indicated on the right.

4370 Recombinant Human Parathyroid Hormone

1 2 3 4 5 6 M

sp

- 100

- 64

1 2 3 4 5 6 7 8 9 1 0 -- “ . . ” - - l o o

4- 64

LJt?”- 45

u- 29

- 45

- 29

- 22

- 12.5

- 6.5

kDa FIG. 3. Extraction of fusion protein. E. coli bacteria harboring

the pEX-PPTH expression plasmid were grown either under nonin- ducing (lanes 1-3) or inducing conditions (lanes 4-6). The cells were harvested, lysed with lysozyme, and extracted with 0 M (lanes 1 and 4 ) , 2 M (lanes 2 and 5 ) , or 9 M urea (lanes 3 and 6 ) . Aliquots of the extracts were electrophoretically analyzed on 17% SDS-polyacryl- amide gels. The arrow points to the position of the fusion protein in lane 6. Marker proteins ( l a n e M) were as in Fig. 2 plus @-galactosidase (100,000).

After optimization of fusion protein synthesis with respect to growth and induction period of the bacteria, the yield of fusion protein obtained from different preparations was be- tween 200 and 300 mg/liter, Le. more than 50% of the total bacterial protein consisted of fusion protein. This corresponds to approximately 35-50 mg of hPTH-sequence/liter culture medium. In comparison, PEW1 optimally yielded 130 mg and pEW2 yielded 50 mg of fusion protein/liter corresponding to approximately 40 and 17 mg of PTH, respectively.

Cleavage of the Fusion Proteins-To hydrolyze the cro-8- galactosidase-PTH fusion protein, the 9 M urea extract was mixed with an equal volume of concentrated formic acid. After 5 days a t 37 “C, the hydrolysis mixture was adjusted to 10 mM ammonium acetate by a stepwise dialysis procedure (see “Experimental Procedures”).

The dialyzed hydrolysis mixture was analyzed by SDS- PAGE (Fig. 4, lane 1 ). A distinct band represents a peptide of approximately 9600 Da, which is the expected molecular mass for Pro-’-hPTH(1-84). This band specifically appeared only after hydrolysis of the Asp-Pro-containing fusion protein but not of an otherwise identical construct lacking this acid- labile linkage (Fig. 4, lane 2). Immunoblotting with anti-PTH antibodies revealed a predominant band of 9.6 kDa (Fig. 4, lanes 4 and 5) as well as several intermediate reaction prod- ucts, most of them appearing only in small quantities (com- pare with lanes 1 and 2).

Attempts to improve the yield of free PTH by extending the cleavage time were not successful as this led to a degra- dation of the hormone itself. Variation of temperature or acid concentration did not improve the yield and no influence of the protein concentration was observed. Use of acetic acid

kDo

- l o o

- 64 - 45

- 29

- 22

- 12.5 - 6.5

kDa

FIG. 4. Release and purification of PTH. Cro-@-galactosidase fusion protein was hydrolyzed by 50% formic acid in 4.5 M urea. After 3 days at 37 “C, the reaction mixture was analyzed by electrophoresis on a 17% SDS polyacrylamide gel ( l a n e 1 ) . For control, another fusion protein similar to that encoded by pEX-PPTH but lacking the proline residue in front of the hPTH sequence was treated identically ( l a n e 2). The gel was blotted to nitrocellulose which was successively incubated with anti-hPTH-antibodies from sheep, rabbit anti-sheep, and goat anti-rabbit antibodies, the latter two being coupled with peroxidase. Immunoreactive bands were visualized by reaction with chloronaphthol and hydrogen peroxide (lanes 4 and 5, corresponding to lanes 1 and 2). The marker was stained with amido black ( l a n e 6 ) . The partially hydrolyzed fusion protein was applied to carboxy- methyl-cellulose at 10 mM ammonium acetate. The unbound peptides were washed away and analyzed by SDS-PAGE ( l a n e 7); bound hPTH was desorbed with 100 mM ammonium acetate ( l a n e 8, 10 pl; lane 9, 20 pl of the eluate). Marker proteins (lanes 3, 6, and 10) were as in Fig. 2.

I

n

I W

II hl U

1 . 2

1 .o

0.8

0.6

0.4

0 . 2

00

80

I

I I v

60 5 .r(

L L 4 C 0 L

40

U

20

time (min) FIG. 5. Purification of recombinant hPTH by HPLC. Recom-

binant hPTH purified by carboxymethyl-cellulose and ammonium sulfate precipitation was subjected to reverse-phase chromatography using a C, column (see “Experimental Procedures” for details). The absorption was recorded at 215 nm. The fraction marked with an asterisk was subjected to rechromatography and was used for amino acid analysis.

Recombinant Human Parathyroid Hormone 4371

instead of formic acid also did not facilitate the hydrolysis. Lower concentrations of urea decreased the hormone yield due to inaccessibility of the cleavage site, whereas the use of guanidine hydrochloride as denaturant led to an accelerated degradation of the PTH itself.

Purification of the Recombinant hPTH-The hydrolysis mixture from either fusion protein which had been dialyzed against 10 mM ammonium acetate was adjusted to 8 M urea. After removal of residual insoluble debris, the solution was loaded onto a carboxymethyl-cellulose column. The unbound peptides were thoroughly removed by extensive washing with

TABLE I Amino acid Composition of purified recombinant human PTH

Amino Residues/ Residues/ Human acid mol” molb Pro”-F’TH

Asx Thr Ser Glx Pro

Ala Val Met Ile Leu Phe His Trp LYS Arg Tyr

G ~ Y

9.9 1.4 6.1 10.9 3.5 4.0 7.1 7.0

1.3 10.2 1.0 4.3

10.2 5.6 0

10.7 2.0 5.5 10.7 4.3 4.1 7.4 7.2 2.0 1.4 9.9 1.1 3.6 1.3 8.5

0 5.4

10 1 7 11 4 4 7 7 2 1

10 1 4 1 9 5 0

“Amino acid composition was analyzed after hydrolysis with 6 N HC1, and the composition was calculated on the basis of 82-amino acid residues (without 2 Met and 1 Trp).

* Hydrolysis was performed with 6 N HCl in the presence of 7% thioglycolic acid, calculation refers to 85-amino acid residues.

8 M urea, 10 mM ammonium acetate until no protein could be detected in the washing buffer (Fig. 4, lane 7). Subsequently, bound peptides were eluted with 8 M urea, 100 mM ammonium acetate. Analysis by SDS-PAGE revealed the selective de- sorption of the recombinant PTH by this protocol (Fig. 4, lanes 8 and 9). The resulting hormone solution was dialyzed against 10 mM acetic acid where it remains stable and biolog- ically active for several months.

Due to incomplete acid hydrolysis as well as to progressive cleavage of the hormone itself and due to severe losses of the product during the several dialysis steps, the final yield of purified PTH was approximately 3-5 mg/liter cell culture. However, regarding the high efficiency of the expression system described above it seems likely that further optimiza- tion of the purification will improve the amounts of product significantly.

On analytical scale, the product obtained after ion exchange chromatography was purified further by reversed-phase HPLC (Fig. 5). Amino acid analysis confirmed the expected composition of the recombinant hormone either in the ab- sence or in the presence of thioglycolic acid during hydrolysis to protect methionine and tryptophane residues (Table I). Sequencing of the NHz-terminal 44-amino acid residues also revealed the authentic sequence including the proline residue in position -1 as well as the asparagine residues at positions 10, 16, and 33 and the glutamine in position 6 and 29 which conceivably could have been deamidated by the acid hydrol- ysis procedure.

Biological Characterization of the Recombinant PTH-The biological activity of the purified recombinant hPTH was assayed by its ability to stimulate renal adenylate cyclase. For this purpose, we used a porcine kidney cortical plasma mem- brane preparation as described (Mohr and Hesch, 1980). Adenylate cyclase has been shown previously to be optimally stimulated by the derivatized PTH fragment [Nle’, Nle”,

0

T

FIG. 6. Biological activity of the recombinant hPTH. The stimulation of porcine renal adenylate cyclase by either the recombinant hPTH (*), native bovine PTH (01, or synthetic hPTH (x) was determined as described under “Experimental Procedures.” Basal activity was substracted from all points. Stimulation of adenylate cyclase by [Nlea,Nle’a,Tyr34]bPTH(1-34) amide at a concentration of 0.92 p M was taken as 100% of control.

4372 Recombinant Human Parathyroid Hormone

T~r~~IbPTH(1-34)amide (Potts et al., 1982). In agreement with published data, we found maximal stimulation by this peptide at 0.92 M which serves as 100% value for the subse- quent experiments.

For comparison, we used either native bovine PTH(1-84) or synthetic human PTH( 1-84). Both reference peptides stim- ulated adenylate cyclase activity with an apparent kact value of 3 nM (Fig. 6). The recombinant Pro-'-hPTH-stimulated CAMP synthesis in a very similar manner as the synthetic hPTH revealing a kaCt value of 6.5 nM (Fig. 6).

Furthermore, we could show that the recombinant hPTH was able to stimulate skeletal adenylate cyclase as well as it enhanced cell proliferation of several target cells very simi- larly to the reference peptides.'

DISCUSSION

We have developed an efficient expression system for hu- man parathyroid hormone by fusing its gene to the cro-lacZ gene of pEX expression vectors. Our first attempts used the complete pEX vector which was supplemented by a short DNA sequence encoding the tetrapeptide Ile-Glu-Gly-Arg; this sequence is known to be the recognition site for blood coagulation factor Xa (Nagai et al., 1985). Downstream from this sequence the human PTH gene was inserted. This con- struct governed highly efficient synthesis of the corresponding fusion protein which, however, was not amenable for factor Xa cleavage even when highly purified protease and fusion protein were a ~ p l i e d . ~ Most likely, the factor Xa recognition sequence within this fusion protein is shielded by extensive secondary or higher structure formation. Secondary structure predictions supported this hypothesis (not shown).

Replacement of the whole sequence between the EcoRV site within the cro-lacZ and the NsiI site within the PTH gene by a synthetic DNA duplex which encodes the native NH2 terminus of hPTH plus an additional proline codon at the P-galactosidase-PTH junction led to a fusion protein which (i) is synthesized even more efficiently (more than 50% of total bacterial protein), (ii) contains a higher portion of PTH sequence, and (iii) contains an acid-labile Asp-Pro link- age between its pro- and eukaryotic moiety. However, further shortening the cro-P-galactosidase part of this hybrid protein did not lead to additional increase in product yield. The most extreme deletion which NHz-terminally to the PTH leaves only 10 amino acids of the cro repressor and some artificial residues resulting from the pEX construction (Zabeau and Stanley, 1982; Stanley and Luzio, 1984), did not lead to either soluble or insoluble minifusion protein detectable by immu- noblot analysis. Although this finding has not been investi- gated further, it coincides with the observation that attempts to express PTH directly in E. coli led to low product levels due to mRNA and protein instability (Breyel et al., 1984; Rabbani et al., 1988; Morelle and Mayer, 1988).

Using either the pEX-PPTH or PEW1 construct, high amounts of PTH-containing fusion protein were expressed. This is readily cleaved by acid hydrolysis and can be purified by a simple two-step procedure involving cation exchange chromatography, which is also suitable for batch procedures, and reversed-phase HPLC mainly for removal of nonprotein- aceous contaminations.

Besides the expected physical characteristics, the recombi- nant hPTH with an additional NH2-terminal proline residue displayed full biological activity when assayed for stimulation of renal cortical plasma membrane in vitro. This was surpris-

* K.-D. Schluter, H. Hellstern, E. Wingender, and H. Mayer, man- uscript submitted.

E. Wingender, unpublished results.

ing as other NHz-terminal modifications, e.g. by an additional Tyr-' residue, have been shown previously to severely affect this hormonal activity (Potts et al., 1982). In agreement with these previous findings, we observed that uncleaved fusion protein was inactive in this assay (data not shown). On the other hand, it might be possible that the Pro" even serves as a protection against attack by aminopeptidases. This would result in prolonged half-time of the hormone thus extending its effect in uiuo.

To assay more specifically for the anabolic activity of PTH, we investigated the increase of cell proliferation of chicken chondrocytes under hormonal influence.' In this test, the recombinant parathyroid hormone described here also exerted full activity when compared with either synthetic human or native bovine PTH(1-84).4

The recombinant material described above provides the basis for numerous structural and functional investigations (in vitro and in vivo) without limitations by the amount of hormone available. Additionally, the expression system pre- sented here is suitable for site-directed mutagenesis to study the individual effects of PTH on the level of single amino acid residues.

Acknowledgments-We want to thank G. Morelle for his help in the initial stages of this work and Dr. G. Gross for stimulating discussions and critically reading this manuscript. The expert tech- nical help of H. Mielke, W. Heikens, and C. Giesa is gratefully acknowledged. We are also indebted to R. Getzlaff and W. Golebski for analyzing the amino acid composition and for sequencing the recombinant PTH.

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