Expression of Human Parathyroid Hormone in ... - Semantic Scholar

THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

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 BiotechnologischeForschung, 0-3300Braunschweig, Federal Republic of Germany

Human parathyroid hormone (PTH) has been expressed in Escherichia coli as a cro-8-galactosidasehPTH fusion protein under temperature-sensitive control of the X phage PRpromoter. 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 linkage between the prokaryotic and the eukaryotic protein moiety consists of an Asp-Pro peptide bond and therefore is easily cleavable by acid treatment. A simple procedure for the purification of the hormone is described. The resulting recombinant hormone reacts with anti-PTH antibodies and stimulates renal adenylate cyclaseidentically 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 mobilization, respectively. Moreover, it also leads to an enhancement of bone remodeling processes (for review see Potts et al., 1982). This has been demonstrated by its ability to stimulate cell proliferation of chondrocytes or osteoblasts in appropriate i n vitro cell systems (van der Plaas et al., 1985; Lewinson andSilbermann, 1986; Kawashima et al., 1980; Burch and Lebovitz, 1983). Thus, parathyroid hormone exhibits differential catabolic as well as anabolic effects. Some recently published studies revealed the promising effect of therapeutical application of PTH as an agent against osteoporosis (Slovik et al., 1987). For this purpose an expression system would be of interest providing sufficient material for further studies and, possibly, clinical applications. Furthermore, such an expression system would provide a basis for protein engineering attempts toinvestigate 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-)constructing 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 “advertisement” in accordance with 18U.S.C. Section 1734 solelyto 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 sulfatepolyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography.

’

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-terminally heterogeneous and required a relatively intricate purification procedure leaving maximally 10 pg/liter culture (Rabbani et al., 1988). To circumvent these problems apparently inherentto direct PTH expression in E. coli, we constructed a series of expression plasmids allowing synthesis of a cro-@-galactosidasehPTH fusion protein. They contain a variable prokaryotic portion anda 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. EXPERIMENTALPROCEDURES

Materials-Restriction enzymes werefrom Boehringer (Mannheim, West Germany) or from GIBCO and Bethesda Research Laboratories. Anti-PTH antibodies were a kind gift from Prof. Hesch, Medizinische Hochschule Hannover. Horseradish peroxidase-conjugated 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 hPTHgene and the EcoRV site of pEX. The resulting plasmid transformed into E. coli N4830. Allcloning procedures were performed according to the standard 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 expression plasmid. If not stated otherwise, this culture was shaken at 30 “C for 4 h; a t 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. Subsequently, 15 ml of 10% sodium deoxycholate were added and after 30 min on ice the mixture was centrifuged (20 min, 8500 rpm ina 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 1h. Centrifugation (20 min, 8500 rpm) yielded another pellet which was resuspended in 10 mlof 9 M urea, 20 mM Tris/HCl, pH 7.4, 2 mM dithiothreitol.

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Hormone Parathyroid Human Recombinant

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After 1 h at room temperature, this suspension was centrifuged (30 min, 9000 rpm). The last step was repeated until virtually all extractable 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 neutralize the formic acid. When the pHof the dialysis bath changed to neutral, dialysis proceededagainst 5 liters of water (withtwo changes) and 10 mM ammonium acetate. Purification of the Recombinant hPTH-The solution was readjusted to 8 M urea and loaded onto acolumn of carboxymethylcellulose (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 ammonium sulfate precipitation (3 M (NH&S04) followed by dialysis against 10 mM aceticacid to removeresidual ureaorsalt.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). Elution 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 according to Bradford (1976) using the Bio-Rad assay with bovine serum albumin as standard. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was as described byLaemmli (1970) on 17% acrylamide, 0.34% bisacrylamide gels. Immunoblotting was essentially asdescribed (Towbin etal., 1979). Binding of anti-PTH antibodies proceeded overnight a t room temperature,binding of peroxidase-conjugated antibodies(rashantisheep andgoat anti-rabbit) for 1 h each. Staining was with I-chloro1-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 of7% thioglycolic acid, analysis of the amino acid composition was performed with an amino acid analyzer LC5001 (Biotronik) connected

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FIG. 1. Expression plasmids for hPTH. In pEX-PPTH, thegene for the human PTH (cross-hatched)which was elongated at its5' end by a proline codon (CCG) was fused to the truncatedcro-lacZ gene which encodes the hybrid protein cro-P-galactosidase(1-407)(hatched). Transcription is under the control of the PRpromoter (black) in the indicateddirection. Starting with thisplasmid, we deleted the HpaI-HpaI, the HpaI-BamHI, or the HindIIIBamHI fragment to obtain the plasmids pEW1, pEW2, or pEW3, respectively. At the bottom, thesequence of the P-galactosidase-hPTH junction isgiven. The DNA sequence shown in italics was chemically synthesized.

Recombinant HumanHormone Parathyroid with a Shimadzu C-R3Aintegrator. Determination of the aminoacid sequence was achievedusinga 470A protein sequencer which is connected on-line with a 120A P T H analyzer (Applied Biosystems). Adeny!ate Cyclase Assay-Porcine renal cortical membranes were prepared and enriched by differential centrifugation and ultracentrifugation on continuous Percoll density gradients by the method of Mohr and Hesch (1980). Adenylate cyclase activity was measured bythe formation of CAMP from ATP according to Mohr and Hesch (1980) with some modifications. Incubation was carried out at30 “C for30 min in a mixture consisting of 50 mM Tris/HCl, pH 7.4, 1.8 mM ATP, 8 mM creatine phosphate, 100 pgof creatine phosphokinase, 1 mM 3-isobutyl-lmethylxanthine, 4.5 mM MgC12,15 mM KCI, 10 p~ Gpp(NH)p, 50 pg of membrane protein, andvariable 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 pEXexpression vectors described by Stanley and Luzio (1984), we inserted the gene for the human PTH between the restriction sites for EcoRV (immediately 3‘ to theAsp(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 ahuman genomic clone (Mayer etal., 1984), taking thesequence 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 aminoacid residues up to theNsiI site was chemically synthesized as a DNA duplex using a previously published method (Frank etal., 1983,1987): 5”CCG T G T GTG AGT GAA ATT CAG CTT ATG CA -3’ 3”GGC ACACAC TCA CTT TAAGTC GAA T -5 ‘

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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 portion (notshown). 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 initially 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.

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Deviating from the original human base sequence, we substituted in this part of the gene the isoleucine 5 codon ATA, which in E. coli is very rarely used (Maruyama et al., 1986), V 29 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 AspPro linkage between the prokaryotic andthe eukaryotic moiety. Thus, acid hydrolysis should release a human para22 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 the8galactosidase-hPTH gene junction was subsequently short12.5 ened by the following in frame deletions: (i) deletion of the HpaI-HpaI fragment removed the amino acid residues 179386 from the cro-P-galactosidase portion (pEW1); (ii) restric6.5 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 kDa fragment after fill-in reaction removed the codons for residues FIG.2. Expression of cro-&galactosidase fusion proteins. 211-406 (pEW3). ml cultures of E. coli harboring theexpression plasmid shown in Fig. Expression-All theconstructs mentioned were trans- 1 were grown for 1 h a t 30 ‘C and 4h at 42‘C. The cells were formed into E. coli strain N4830 harboring a temperatureharvested, the proteins solubilized with 1% SDS and analyzed by sensitive X repressor in its genome (cI857) (Gottesman et al., electrophoresis on a 17% SDS-polyacrylamide gel. The positions of 1980). Therefore, growth at 30 “Ccompletely represses the X theinduction-dependent fusion proteins encoded by pEX-PPTH PRpromoter but allows induction of the PR-controlled gene ( l a n e 2), PEW1 (lane 3), and pEW2 (lane 4 ) are indicated by the arrows on theleft. The protein patternof noninduced control cells (5 by temperature shift to42 “C. h a t 30 “C) is shown as well ( l a n e 1 ). Marker proteins ( l a n e M )are Total protein extracts of small-scale cultures of pEX- bovine serum albumin, ovalbumin, carboanhydrase, trypsin inhibitor PPTH, pEW1, and pEW2 displayed very clear induction- from soybean, cytochrome c, and trypsin inhibitor from lung. The specific bands when analyzed on SDS-polyacrylamide gel molecular masses are indicated on the right.

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FIG.3. Extraction of fusion protein. E. coli bacteria harboring the pEX-PPTHexpression plasmid were grown either under noninducing (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-polyacrylamide gels. The arrow points to theposition of the fusion protein in lane 6. Marker proteins ( l a n e M )were as in Fig. 2 plus @-galactosidase

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FIG.4. Release and purification of PTH. Cro-@-galactosidase fusion protein was hydrolyzedby 50% formic acid in 4.5 M urea. After 3 days a t 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 thatencoded by pEX-PPTH but lacking the proline residue in frontof the hPTHsequence 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 carboxymethyl-cellulose a t 10 mM ammonium acetate. The unbound peptides were washed awayand 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.

(100,000).

After optimization of fusion protein synthesis withrespect to growth and induction period of the bacteria, the yield of fusion protein obtained from different preparations was between 200 and 300 mg/liter, Le. more than 50% of the total bacterial proteinconsisted 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-8galactosidase-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 SDSPAGE (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 acidlabile 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 products, most of them appearing only in small quantities (compare 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 degradation of the hormone itself. Variation of temperature oracid concentration did not improve the yield and no influence of the protein concentration was observed. Use of acetic acid

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time (min) FIG.5. Purification of recombinant hPTH by HPLC. Recombinant 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 a t 215 nm. The fraction marked with an asterisk was subjected to rechromatography and was used for amino acid analysis.

Recombinant H u m a n Parathyroid Hormone 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 acarboxymethyl-cellulose column. The unbound peptides were thoroughly removed by extensive washing with TABLEI Amino acid Composition of purified recombinanthuman PTH Amino acid

Residues/ mol”

Residues/ molb

Human Pro”-F’TH

10 Asx 10.7 9.9 1 2.0 Thr 1.4 7 5.5 6.1 Ser 11 10.7 10.9 Glx 4 4.3 Pro 3.5 4 4.1 4.0 G~Y 7 Ala 7.4 7.1 7 7.2 Val 7.0 2 2.0 Met 1 1.4 Ile 1.3 9.9 10 Leu 10.2 1.1 1 Phe 1.0 4 3.6 His 4.3 1 1.3 Trp 8.5 9 10.2 LYS 5.4 5 5.6 Arg 0 0 0 Tyr “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.

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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 desorption of the recombinant PTH by this protocol (Fig. 4, lanes 8 and 9). The resulting hormone solution was dialyzed against 10mM acetic acid where it remains stable andbiologically 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 highefficiency of the expression system described above it seems likely that further optimization 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 absence or in thepresence 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 hydrolysis 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 membranepreparationas described (Mohrand Hesch, 1980). Adenylate cyclase has been shown previously to be optimally stimulated by the derivatized PTH fragment [Nle’, Nle”,

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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 amide at a concentration of 0.92 p M was taken as 100% of control. by [Nlea,Nle’a,Tyr34]bPTH(1-34)

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Recombinant HumanHormone Parathyroid

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 subsequent experiments. For comparison, we used either native bovine PTH(1-84) or synthetic human PTH( 1-84). Both reference peptides stimulated 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 kaCtvalue 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 similarly to thereference peptides.'

ing as otherNHz-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 thePro" 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 specificallyfor 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 DISCUSSION The recombinant material described above provides the basis for numerous structural and functional investigations We have developed an efficient expression system for human parathyroid hormone by fusing its gene to the cro-lacZ (in vitro and in vivo) without limitations by the amount of gene of pEX expression vectors. Our first attempts used the hormone available. Additionally, the expression system presented here is suitable for site-directed mutagenesis to study complete pEX vector which was supplemented by a short DNA sequence encoding the tetrapeptide Ile-Glu-Gly-Arg; the individual effects of PTH on the level of single amino this sequence is known to be the recognition site for blood acid residues. coagulation factor Xa (Nagai et al., 1985). Downstream from Acknowledgments-We want to thank G. Morelle for his help in this sequence the human PTH gene was inserted. This con- the initial stages of this work and Dr. G. Gross for stimulating struct governed highlyefficient synthesis of the corresponding discussions and critically reading this manuscript. The expert techfusion protein which, however, was not amenable for factor nical help of H. Mielke, W. Heikens, and C. Giesa is gratefully Xa cleavage even when highly purified protease and fusion acknowledged. We are also indebted to R. Getzlaff and W. Golebski protein were a ~ p l i e dMost . ~ likely, the factor Xa recognition for analyzing the amino acid composition and for sequencing the sequence within this fusion protein is shielded by extensive recombinant PTH. secondary or higher structure formation. Secondary structure REFERENCES predictions supported this hypothesis (not shown). Bradford, M. M. (1976) Anal. Biochem. 7 2 , 248-254 Replacement of the whole sequence between the EcoRV Breyel, E., Morelle, G., Aufmkolk, B., Frank, R., Blocker, H., and site within the cro-lacZ and the NsiI site within the PTH Mayer, H. (1984) 3rd European Congress on Biotechnology 3 , 363gene by a synthetic DNA duplex which encodes the native 369 NH2 terminus of hPTH plus an additional proline codon at Broker, M. (1986) Gene Anal. Techn. 3,53-57 the P-galactosidase-PTH junction led to a fusion protein Burch, W. M., and Lebovitz, H. E. (1983) Calcif. Tissue Znt. 3 5 , 526532 which (i) is synthesized even more efficiently (more than 50% Frank, R., Heikens, W., Heisterberg-Moutsis, G., and Blocker, H. of total bacterial protein), (ii) contains a higher portion of (1983) Nucleic Acids Res. 11,4365-4377 PTH sequence, and (iii) contains an acid-labile Asp-Pro link- Frank, R., Meyerhans, A., Schwellnus, K., and Blijcker, H. (1987) age between its pro- andeukaryotic moiety. However, further Methods Enzymol. 1 5 4 , 221-249 shortening the cro-P-galactosidase part of this hybrid protein Gottesman, M. E., Adhya, S., and Das, A. (1980) J. Mol. Biol. 140, 57-75 did not lead to additional increase in product yield. The most Kawashima, K., Iwata, S., and Endo, H. (1980) Endocrinol. Jpn. 2 7 , extreme deletion which NHz-terminally to the PTH leaves 349-356 only 10 amino acids of the cro repressor and some artificial Keutmann, H. T., Barling, P. M., Hendy, G. N., Segre, G. V., Niall, residues resulting from the pEX construction (Zabeau and H. D., Aurbach, G. D., Potts, J. T., Jr., and O'Riordan, J. L. H. Stanley, 1982; Stanley andLuzio, 1984),did not lead to either (1974) Biochemistry 1 3 , 1646-1652 soluble or insoluble minifusion protein detectable by immu- Laemmli, U. K. (1970) Nature 227,680-685 noblot analysis. Although this finding has not been investi- Landon, M. (1977) Methods Enzymol. 1 5 4 , 145-149 gated further, it coincides with the observation that attempts Lewinson, D.,and Silbermann, M. (1986) Cakif. Tissue Int.3 8 , 155162 to express PTH directly in E. coli led to low product levels Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular due to mRNA and protein instability (Breyel et al., 1984; Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York Rabbani et al., 1988; Morelleand Mayer, 1988). Using either the pEX-PPTH or PEW1 construct, high Marston, F. A. 0.(1986) Biochem. J. 2 4 0 , 1-12 amounts of PTH-containing fusion protein were expressed. Maruyama, T., Gojobori, T., Aota, S.-i., and Ikemura, T. (1986) Nucleic Acids Res. 1 4 , r151-rl97 This is readily cleaved by acidhydrolysis and can be purified Mayer, H., Breyel, E., Bostock, C., and Schmidtke, J. (1983) Human by a simple two-step procedure involving cation exchange Genet. 64,283-285 chromatography, which is also suitable for batch procedures, Mayer, H., Widera, G., Breyel, E., and Schmidtke, J. (1984) ICSCJ and reversed-phase HPLC mainly for removal of nonproteinShort Reports 1 , 206-207 Mohr, H., and Hesch, R.-D. (1980) Biochem. J. 188,649-656 aceous contaminations. Besides the expected physical characteristics, the recombi- Morelle, G., and Mayer, H. (1988) Biochim. Biophys. Acta 9 5 0 , 459462 nant hPTH with an additional NH2-terminal proline residue Nagai, K., Perutz, M. F., and Poyart, C. (1985) Proc. Natl. Acad. Sci. displayed full biological activity when assayed for stimulation U. S. A. 82,7252-7255 of renal cortical plasma membrane in vitro. This was surpris- Potts,J. T., Kronenberg, H. M., and Rosenblatt, M. (1982) Adu.

* K.-D. Schluter, H. Hellstern, E. Wingender, and H. Mayer, manuscript submitted. E. Wingender, unpublished results.

Protein Chem. 3 2 , 323-395 K.-D. Schuter, G. Bercz, E. Wingender, and H. Mayer, manuscript in preparation.

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