ARCHIVES
Vol.
OF BIOCHEMISTRY
294, No. 2, May
AND
BIOPHYSICS
1, pp. 579-585,
1992
Purification and Characterization of Recombinant Porcine Prorelaxin Expressed in Escherichia co/? G. Kesava Reddy,* Anthony B. Chen,t
Sripad Gunwar,* Carla B. Green,**2 and Simon C. M. Kwok*p3
David
T. W. Fei,?
*Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160; and tDepartment of Medicinal and Analytical Chemistry, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, California 94080
Received
October
24,1991,
and in revised
form
January
13,1992
In th$ report we describe the purification and characterization of recombinant porcine prorelaxin expressed in Eecherichia coli. Nucleotide sequence encoding porcine prorelaxin was inserted into an E. coli expression vector, POTS, and the recombinant plasmid was transformed into the E. coli host (AB120). Upon induction with nalidixic acid, the 19-kDa recombinant porcine prorelaxin was produced at a level of approximately 8% of the total accumulated cell protein. The recombinant prorelaxin was purified to homogeneity by CM-cellulose chromatography and reversed-phase HPLC, after refolding in the presence of reduced and oxidized glutathione and a low concentration of guanidine-HCl. The identity of the recombinant prorelaxin was confirmed by the correct size, immunoreactivity with antibodies against native porcine relaxin, and direct amino-terminal sequence analysis. Furthermore, the purified recombinant prorelaxin could be converted to the 6-kDa relaxin by limited digestion with trypsin. Trypsin was shown to cleave at the carboxyl side of Arg29 and Arg’37 residues of the recombinant prorelaxin, producing the des-A&‘-B29-relaxin, and degrade the 13-kDa connecting peptide into small peptides. Both the recombinant prorelaxin and converted relaxin were found to be biologically active in an in vitro bioassay for relaxin. Q 1992 Academic Press, Inc.
Relaxin is a 6-kDa peptide hormone which is composed of two peptide chains (A- and B-chains) linked by two interchain disulfide bonds. It is primarily synthesized by the corpus luteum during pregnancy, although a low level ‘This work was supported by U.S. Public Health Service HD24599. ’ Present address: Department of Anatomy and Cell Biology, versity of Kansas Medical Center, Kansas City, KS 66160. 3 To whom correspondence should be addressed. OOO3-9861/92 $3.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
Inc. reserved.
Grant Uni-
of relaxin activity has also been detected in the nonpregnant ovary, uterus, placenta, testes, and prostate (1, 2). The hormone exerts a profound effect on the reproductive tract during pregnancy. Its major biological functions are: (a) ripening of the uterine cervix, (b) relaxation of the pubic symphysis to facilitate birth, (c) inhibition of uterine contraction to prevent premature birth, and (d) development of the mammary gland. The amino acid sequences of relaxin have been determined by direct protein sequencing or deduced from the nucleotide sequences of the cDNAs for a number of species including pig (3-6), rat (7, 8), sand tiger shark (9), human (lo), spiny dogfish (ll), skate (121, whale (131, and monkey (14). Nucleotide sequence analysis of the relaxin cDNAs revealed that relaxin is synthesized as a single-chain 23-kDa preprorelaxin with the overall structure: signal peptide, B-chain, connecting peptide, and A-chain. During the biosynthesis of relaxin, the signal peptide is removed rapidly as the nascent peptide chain is translocated across the endoplasmic reticulum, producing the 19-kDa prorelaxin. Further processing of the prohormone into the 6-kDa relaxin is not well understood. In order to study the mechanism of conversion of prorelaxin to relaxin, a highly purified preparation of the prohormone is required. Although high molecular weight intermediates have been isolated from porcine ovaries (E), attempts to purify the prohormone from porcine ovaries have not been successful due to its extreme heterogeneity (unpublished result). We have, therefore, decided to produce porcine prorelaxin in Escherichia coli. We report herein the purification and characterization of the recombinant prorelaxin. MATERIALS
AND
METHODS
Bacterial strains and plasmids. E. coli strains (AR120, MM294cI+) and the E. coli expression vector (POTS) are lcmdly provided by Dr. Martin Rosenberg of the Smith Kline & French L&oratories. A plasmid, pPR 308-6, harboring a full-length porcine preprorelaxin cDNA was 579
580
REDDY
isolated from a porcine ovary cDNA library using a 5’-end-labeled oligonucleotide (21-mer) probe (kindly provided by Dr. Donald F. Steiner), which was synthesized based on the published nucleotide sequence of porcine preprorelaxin cDNA (6). The identity of the cDNA was confirmed by restriction endonuclease mapping and nucleotide sequence analysis using the chain termination method (16, 17). A &!&base-pair Construction of the prorekxin expression plasmid. RsaI/EcoRI fragment containing the coding sequence for promlaxm was isolated from plasmid pPR308-6 using a 5% polyacrylamide gel and was inserted into the expression vector, POTS (18), using standard recombinant DNA techniques (19), as depicted in Fig. 1A. The isolated fragment was blunt-ended with a Klenow fill-in reaction and ligated to a synthetic BamHI linker (8-mer). The BamHI-linkered fragment was then subcloned into the BamHI site of the POTS expression vector. The resulting recombinant plasmid was first transformed into E. coli MM294cP. Minipreparations of DNA were prepared as described by Del Sal et ol. (17) for confirmation of orientation by XhoI/BglII digestion and by nucleotide sequence analysis using a sequence-specific primer (16,17). Finally, the recombinant plasmid was transformed into E. coli AR120 for nalidixic acid induction. The clone was designated pPR754AR120. Growth and induction of bacteria Growth and induction of pPR754AR120 was carried out essentially as described by Mott et al. (20). Cultures were grown in maximal induction medium [3.2% tryptone, 2% yeast extract, 1X M9 salts, 0.1 mM MgSO,, 0.001 mM FeCl,, and 100 &g/ml ampicillin (20)] at 37’C until Am reached 1.0 and were then induced with nalidixic acid at a final concentration of 40 fig/ml for 16 h at 37’C. The 16-h induction was chosen as a matter of convenience, since the expression reached a maximum after 5 h of induction and the expressed protein is stable even after 21 h of induction (20). Isolation andpurification of recombinuntproreluxin. The E. coli cells grown and induced as described above were harvested by centrifugation and lysed with lysozyme and deoxycholic acid, and the inclusion bodies were washed with buffer containing 0.5% Triton X-100 and 10 mM EDTA’ as described by Marston (21). Subsequent steps for the isolation of the recombinant prorelaxm were adapted from the procedure described by Winkler and Blaber (22). The washed inclusion bodies were purifled by centrifugation through a 50% glycerol cushion and then solubilixed by 6 M guanidine-HCl in 50 mM Tris-HCl (pH 8.0) containing 100 mM NaCl and 1 mM EDTA (1 ml/g cells) at 4°C for 16 h. Insoluble material was removed by centrifugation. The supernatant was initially diluted with 5 vol of 50 mu Tris-HCl (pH 9.0) containing 5 mM EDTA and then diluted further to Auu, < 1.0 with 1 M guanidine-HCI in the same buffer. The extracted proteins were allowed to refold at 4’C for 24 h in the presence of 0.2 M arginine, 0.005% Tween 80, 1.25 mu reduced glutathione, and 0.25 mM oxidized glutathione (22). The refolded proteins were then dialysed against 10 mu ammonium acetate (pH 5.5) containing 1 mM EDTA and 20 pg/ml PMSF, and insoluble material was removed by centrifugation. The clarified protein sample was passed through a 2.5 X 20-cm column of CM-cell~ose (CM-52, Whatman) preequilibrated with 10 mM ammonium acetate (pH 5.5) containing 1 mM EDTA and 20 pg/ml PMSF (starting buffer), and unabsorbed proteins were washed with the same buffer. The absorbed proteins were eluted with a linear gradient of O0.2 M NaCl formed by 300 ml of starting buffer and 300 ml of 0.2 M NaCl in starting buffer. Fractions of 2.5 ml were collected at a flow rate of 15 ml/h. Aliquots of the fractions were analyzed on a 15% SDSpolyacrylamide gel (24), followed by Western blot analysis (described below). Prorelaxin containing fractions were pooled, dialyzed against 10 mM ammonium acetate (pH 5.5) containing 1 mM EDTA and 20 pg/ ml PMSF, and then lyophilized.
’ Abbreviations used: EDTA, ethylenediaminetetraaceta~, PMSF, phenylmethylsulfonyl fluoride; HPLC, high-performance liquid chromatography; SDS, sodium dodecyl sulfate; DFP, diisopropyl fluorophosphate; TPCK, r.,-l-p-tosylamino-2-phenylethyl chloromethyl ketone.
ET
AL.
Subsequent purification of recombinant prorelaxin was carried out by reversed-phase HPLC using a Vydac Cn column. The column was equilibrated with 30% acetonitrile and 0.1% tritluoroacetic acid. Approximately 2 mg of partially purified recombinant prorelaxin from the CM-cellulose column was dissolved in 1 ml of 50 mu Tris-HCI (pH 7.5), adjusted to 0.5% trifluoroacetic acid, and injected into the column. A 30-min gradient of 30-70% acetonitrile was used to elute the proteins at a flow rate of 2 ml/min. Fractions were collected manually for various
A l?zoRI
I!coRI
1
;~~l~+ Eco RI digestion
I
I
Klemw fill-in Barn HI linkers Barn tll dtgestion
Barn HI digestion Dcphnsphmylation
BlUtipd
1
Ligation
B&I HI
B
:zoFrtion
~tor.ldn I
Met
Thr Asn Asp ACG AAC CAT...
AT-
Asp
Pro
*
a&mar linker
FIG. 1. (A) Construction of the prorelaxin expression plasmid pPR754-AR120. The details of the construction are described under Materials and Methods. The open box represents the coding sequence of prorelaxin with the hatched region indicating the coding sequence for the prepeptide. The closed box represents the sequence derived from phage X. 9JPi,, bacteriophage lambda operator and promoter; cI1 r.b.s., cl1 ribosome binding site; to,lambda phage terminator; Amp’, ampicillin resistance. (B) Coding sequence present in pPR754-AR120 at the junction between the prorelaxin and the vector. The initiation codon is provided by the POTS expression vector. The codons for the second and third residues (Asp-Pro) are contributed by the linker (shown in italics). The remaining coding sequence is derived from the porcine prorelaxin cDNA.
RECOMBINANT
PORCINE
peaks and lyophilized immediately. The peak corresponding binant prorelaxin was identified by SDS-polyacrylamide phoresis and Western blot analysis.
PRORELAXIN to recomgel electro-
Limited digestion of recombinant prorelaxin with trypsin. TPCKtrypsin (Sigma) was dissolved in 0.001 N HCl at a concentration of 1 mg/ml. Purified recombinant prorelaxin was dissolved in water at a concentration of 5 pg/Fl. Aliquots of 5 pg of recombinant pmrelaxin were pipetted into a series of microcentrifuge tubes containing 20 /.d of 50 mM Tris-HCl (pH 8.1) and 11.5 mM CaClx. TPCK-trypsin was then added at different enzyme-to-substrate ratios (1:lOO to 1:500 w/w), and the reaction mixture was vortexed briefly and incubated for 5 min at 37’C. The trypsin activity was arrested by freezing on dry ice and the samples were dried in a vacuum evaporator. The tryptic peptides were analyzed by urea-SDS-polyacrylamide gel electrophoresis (25). To isolate the converted relaxin from limited tryptic digest of recombinant pmrelaxin, the digested material was acidified with trifluomacetic acid (to a final concentration of 0.5%) and injected into the Vydac Cis column equilibrated with 10% acetonitrile. A 50-min gradient of lo50% acetonitrile was used to elute the converted relaxin at a flow rate of 1 ml/min. Fractions were collected manually for each peak and lyophilized immediateiy. The converted relaxin peak was identified by urea-SDS-polyacrylamide gel electmphoresis and Western blot analysis. Western blot analysis. The procedure of Bumette (26) was used with minor modifications. The separated proteins were electrophoretically transferred to nitrocellulose, blocked with 5% nonfat dry milk, and reacted with antiserum against porcine relaxin (kindly provided by Dr. Gillian Bryant-Greenwood). After thorough washing, the blot was incubated with a mixture of alkaline phosphatase-conjugated anti-rabbit IgG (Sigma) and streptavidin-alkaline phosphatase conjugate (Pierce). The pmrelaxin/relaxin bands and biotinylated molecular weight markers were then visualized with chromogenic substrates (nitro blue tetrazonium and 5-bromo-4-chloro-3-indolyl phosphate). Carboxypeptidase digestion. The HPLC-purified converted relaxin (20 pg) was digested with DFP-treated carboxypeptidase A or B (Sigma) as described by Schwabe et al. (13). The amino acid(s) released was determined by amino acid analysis. A reagent blank consisting of buffer and enzyme but no relaxin was used to subtract the background.
EXPRESSED
IN
Escherichia
581
coli
residues (Gln-Ser) at the amino terminus replaced by Met-Asp-Pro residues (Fig. 1B). The Met codon was provided by the expression vector, and the Asp-Pro codons were furnished by the BamHI linkers. Purification and characterization of recombinant prorelaxin. Although the recombinant prorelaxin represents 8% of the total E. coli proteins, we took advantage of the fact that prorelaxin forms aggregates known as inclusion bodies. The bulk of bacterial proteins were removed by purifying the inclusion bodies before the solubilization of the recombinant prorelaxin. Other bacterial proteins were removed as precipitates when the refolded proteins were dialyzed against buffer containing no denaturing agents. Further removal of bacterial proteins was accomplished with CM-cellulose chromatography, which also served to concentrate the recombinant prorelaxin from a large volume of diluted protein sample (2.1 liters). Only one major absorbed protein peak was eluted from the CM-cellulose column (data not shown). Western blot analysis showed that this peak contained the recombinant prorelaxin (data not shown). Further purification by reversed-phase HPLC revealed that this partially purified recombinant prorelaxin was composed of three major peaks (designated A, B, and C in Fig. 2). Western blot analysis showed that only peak B, which was eluted at 41% acetonitrile, contained the recombinant prorelaxin (data not shown). The yield of prorelaxin from a 3-liter culture was 2.4-3.0 mg. The results from SDS-polyacrylamide gel electrophoresis suggested that recombinant prorelaxin was purified to homogeneity by two chromatographic steps (Fig. 3, lane
Determination of amino-terminal sequence and amino acid composition. The amino-terminal sequence and amino acid composition analyses of purified recombinant prorelaxin and converted relaxin were performed by Dr. Audree Fowler at the University of California, Protein Microsequencing Facility, Los Angeles, California.
r
Bioassay. The biological activities of recombinant prorelaxin and converted relaxin were determined with the in vitro bioassay using human endometrial cells as described by Fei et al. (27). The concentration of recombinant prorelaxin and converted relaxin were determined spectrophotometrically using the values of Alx,, of 20.5 and 2.0, respectively. The AIZm, value of prorelaxin was determined by amino acid analysis of an aliquot of a sample of known absorbance at 280 nm and that of relaxin was reported earlier (28).
RESULTS
Corzstruction of the prorehxin expression plasmid. A plasmid harboring a full-length porcine preprorelaxin cDNA was used as starting material for the construction of the prorelaxin expression plasmid. In order to express porcine prorelaxin in E. coli, the coding sequence for the prepeptide was removed by RsaI digestion, which also removed the first two codons of the B-chain. The truncated prorelaxin cDNA was then subcloned into the BamHI site of the POTS expression vector using BarnHI linkers (Fig. 1A). This expression plasmid thus produced a variant form of porcine prorelaxin with its first two “authentic”
: I
(
I
I
38 40 42 %ACETONITRILE FIG.
I
I
44
2. Purification of recombinant prorelaxin by reversed-phase HPLC. Partially purified sample from the CM-cellulose column (2 mg) was applied onto the Vydac Cis column and eluted with a linear gradient of acetonitrile as described under Materials and Methods. Three major peaks were obtained in the elution profile. Peak B, which was eluted at 41% acetonitrile, contained recombinant prorelaxin as confirmed by Western blot and amino-terminal sequence analyses.
582
REDDY MW
1
2
3
4
5
6
FIG. 3. Analysis of the products released by limited trypsin digestion of recombinant prorelaxin on urea-SDS-polyacrylamide gel. About 5 pg of recombinant prorelaxin was digested with various concentrations of TPCK-trypsin at 37°C for 5 min, lyophilized, boiled in sample buffer containing no P-mercaptoethanol, and analyzed on a urea-SDS-polyacrylamide gel. The enzyme to substrate ratios (w/w) are as follows: lane 1, 1:lOO; lane 2, 1:200, lane 3, 1:300; lane 4, 1:400; lane 5, 1:500; and lane 6, without enzyme. The molecular weights of 19 and 6 kDa marked the migration of intact recombinant prorelaxin and insulin, respectively, which were also electrophoresed in the absence of &mercaptoethanol.
6). Amino-terminal sequence analysis of the purified recombinant prorelaxin yielded the following sequence: Met -Asp -Pro -Thr -Asn- Asp- Phe- Ile- Lys- Ala -CysGly-Arg-Glu-Leu-Val-Arg-Leu-Trp. This sequence agreed perfectly with that expected from vector construction and the published sequence deduced from porcine preprorelaxin cDNA (6). The first two residues (Gln-Ser) in the natural prorelaxin were replaced by Met-Asp-Pro at the amino-terminal end of recombinant prorelaxin. The amino-terminal Met residue of recombinant prorelaxin is neither blocked by a formyl group nor removed. Limited tryptic digestion. The results from limited tryptic digestion studies indicated that the recombinant prorelaxin could be converted by trypsin to a polypeptide of the size of relaxin. At low concentrations of trypsin, recombinant prorelaxin was partially cleaved, giving rise to a band with slower mobility than the intact prohormone (Fig. 3, lanes 4 and 5). The conversion of recombinant prorelaxin to relaxin occurred gradually at a 1:300 (w/w) enzyme-to-substrate ratio, as visualized by the appearance of a band corresponding to the 6-kDa relaxin on ureaSDS-polyacrylamide gel electrophoresis (Fig. 3, lane 3). At a 1:lOO (w/w) enzyme-to-substrate ratio, only the conversion product of the size of relaxin can be observed during the 5-min incubation period at 37°C (Fig. 3, lane 1). When this tryptic digest was chromatographed on the Vydac Cis column, one major peak was eluted at 39% acetonitrile (Fig. 4). Amino-terminal sequence analysis of the purified conversion product revealed two residues per cycle in approximately equimolar amounts. The results, when compared with the sequence of the B-chain (stated above), yielded the following sequences: Met-Asp-
ET
AL.
Pro-Thr-Asn-Asp-Phe-Ile-Lys-Ala for the B-chain and Met-Thr-Leu-Ser-Glu-Lys-Cys-Cys-Gln-Val for the A-chain. The latter agrees perfectly with the amino-terminal sequence of the A-chain of the native relaxin (3, 4), except that the Ar$l residue in the native relaxin is missing in the converted relaxin. Amino acid analysis of the HPLC-purified, converted relaxin after digestion with carboxypeptidase B revealed the release of 186 pmol of arginine. However, a similar experiment with carboxypeptidase A failed to produce any significant amount of released amino acids above background. Both the recombinant prorelaxin and the converted relaxin were found to be biologically active in an in vitro bioassay for relaxin, with 1 maximal activities (ED& of 11.7 and 6.2 nM, respectively (Fig. 5). During these studies we also examined the stability of the converted relaxin and the fate of the connecting peptide by the action of trypsin following a lo-, 20-, and 30min incubation period with the enzyme-to-substrate ratio kept constant (1:lOO). The results indicated that the relaxin band was observed in samples incubated at lo- and 20-min intervals. However, no relaxin band was detected on the urea-SDS-polyacrylamide gel if the incubation period was over 30 min (data not shown). On the other hand, the expected 13-kDa connecting peptide was not observed even after 5 min of incubation. -
12
20
26
36
44
% ACETONITRILE
FIG. 4. Separation of converted relaxin from limited tryptic digest of recombinant prorelaxin by reversed-phase HPLC. Limited tryptic digest of recombinant prorelaxin (1 mg) was applied onto the Vydac C18 column and eluted with an acetonitrile gradient as described under Materials and Methods. The single major protein peak, which was eluted at 39% acetonitrile, was found to be converted relaxin by Western blot and amino-terminal sequence analyses. The arrow designates position where recombinant prorelaxin (PR) elutes.
RECOMBINANT
PORCINE
100 Porcine
PRORELAXIN
10' Proreloxin
102 CnMI
-f-
L
10-l
I
I
100
I
10' Porcine
Reloxin
102 Cd.4
1
FIG. 5. In vitro bioassay of recombinant prorelaxin and trypsin-convetid relaxin. Human uterine endometrial cells were treated with various dilutions of recombinant prorelaxin (top panel) or converted relaxin (bottom panel), and the amounts of intracellular CAMP produced were determined by radioimmunoassay. Each value is the mean f SD of triplicate determinations. Since maximal CAMP response varies in different batches of cells, the biological activities were expressed as $ maximal activities (ED,), which were found to be 11.7 and 6.2 nM for recombinant prorelaxin and converted relaxin, respectively.
DISCUSSION In the present paper we have described the purification and characterization of recombinant prorelaxin produced by E. coli. The identity of prorelaxin was confirmed by the correct size, immunoreactivity with the antibodies against native porcine relaxin, direct amino-terminal sequence analysis, and biological activity in an in vitro
EXPRESSED
IN
Escherichia
coli
583
bioassay. Furthermore, we have also demonstrated the conversion of the recombinant prorelaxin to relaxin by a limited digestion with TPCK-trypsin. Expression of porcine prorelaxin in E. coli has also been reported by another group of investigators (29). However, to the best of OUI knowledge, no report has been made on the purification and characterization of the recombinant prorelaxin. During the purification of recombinant prorelaxin, we have attempted several procedures. The procedure described in this paper was an adaptation of the method of Winkler and Blaber (22) and was used with success. The procedure involves extraction of the recombinant prorelaxin with 6 M guanidine-HCl, followed by refolding of the crude extract in the presence of oxidized and reduced glutathione and 1 M guanidine-HCl. By refolding the recombinant prorelaxin in the presence of oxidized and reduced glutathione prior to chromatography, the use of denaturing agents can be eliminated in the subsequent purification procedures. Furthermore, the recombinant prorelaxin can be purified to homogeneity by merely two chromatographic steps. In the other procedure, the recombinant prorelaxin was converted into an S-sulfonate derivative by the reaction with sodium sulfite and sodium tetrathionate as described by Cabilly et al. (30). The Ssulfonated recombinant prorelaxin was then subjected to gel filtration and ion-exchange chromatography using buffers containing denaturing agents. This procedure proved to be unsuccessful for the purification of recombinant prorelaxin. The immunoreactive materials appeared as a smear in the chromatographic profiles (data not shown). In this report we use TPCK-trypsin as a model enzyme to study the conversion of recombinant porcine prorelaxin and to establish a “conversion assay” for the prorelaxin processing protease( Although trypsin is not the authentic prorelaxin processing protease, it has been used successfully to convert high molecular weight forms of porcine relaxin to the 6-kDa relaxin (15). Trypsin can easily cleave at the carboxyl side of the cluster of four basic residues (residues 131-134) near the connecting peptide/A-chain junction (6). On the other hand, although the processing enzyme for the B-chain/connecting peptide junction is not a trypsin-like enzyme, trypsin may cleave at the carboxyl side of Arg2’, which is located only three residues away on the amino side of the proposed cleavage site (6). Our results on limited digestion of recombinant prorelaxin with trypsin showed that it can be converted to the 6-kDa relaxin. Amino-terminal sequence analysis of the converted relaxin reveals that the Arg residue at the amino terminus of the A-chain is removed. This suggests that prorelaxin is cleaved at the carboxyl side of Arg137 instead of the cluster of four basic residues (positions 131-134) near the connecting peptide/A-chain junction (6). On the other hand, cleavage at the B-chain/ connecting peptide junction was inferred indirectly from the experiments with carboxypeptidases A and B. Since
584
REDDY
the carboxyl-terminal Cys residue of the A-chain is involved in the formation of a disulfide bond, carboxypeptidases A and B will only release amino acids from the carboxyl terminus of the B-chain. Carboxypeptidase B digestion of the converted relaxin released only arginine, while carboxypeptidase A did not release any amino acids. The results are consistent with the prediction that the cleavage may occur between Arp and Th$O, leaving Arp as the carboxyl terminus of the B-chain. The converted relaxin is thus des-Are’-B29-relaxin. Both the recombinant prorelaxin and converted relaxin were found to be biologically active in the in vitro bioassay for relaxin. However, whether the biological activity of the recombinant prorelaxin is due to its intrinsic property or on-site conversion on the surface of target cells remains to be determined. Conversion with trypsin increased the biological activity of relaxin by merely twofold. Although earlier observations have shown that high molecular weight forms of relaxin are biologically active (15), the present study unequivocally demonstrates for the first time that prorelaxin is biologically active. On the other hand, the biological activity of the converted relaxin is about threefold less than that of the native porcine relaxin, which has a EDso value of 1.98 nM (27). The discrepancy in the biological activities of the recombinant and native relaxins may be explained by several structural differences. First, the recombinant relaxin differs slightly from the native relaxin in that it contains Met-Asp-Pro residues instead of Gln-Ser residues at the amino terminus of the B-chain as a result of the expression plasmid construction. Second, not all the recombinant relaxin molecules may be refolded correctly. Third, the removal of Arg at the amino terminus of the A-chain and the Thr30-Ala31-Leu32 residues at the carboxyl terminus of the B-chain may lead to the lower biological activity of the converted relaxin in the in vitro bioassay. However, it is interesting to note that the removal of Thr3’-Ala31Leu32 at the carboxyl terminus of the B-chain will generate a molecule corresponding to the B29 relaxin (CM-a’) which possesses full biological activities in the in vivo bioassay (31,32) and that the removal of the Arg residue at the amino terminus of the A-chain has little effect on the biological activity of the relaxin, also in the in vivo bioassay (33). We also looked at the fate of the connecting peptide, as it is better conserved than the A- and B-chains in relaxins of different species studied thus far and hence may possess a biological function(s) other than folding the two chains into proper conformation. In our experiments, we found that at a 1:lOO (w/w) enzyme-to-substrate ratio, recombinant prorelaxin is optimally converted to relaxin during a 5-min incubation period at 37°C. The converted relaxin is stable under these conditions for up to 20 min of incubation, but is completely degraded during longer incubation. However, even after 5 min of incubation, we are unable to detect the expected 13-kDa connecting pep-
Fe.1 --
AL. AT
tide on the SDS-polyacrylamide gel. A closer examination on the amino acid sequence of the porcine prorelaxin connecting peptide (6) reveals at least 16 possible cleavage sites for trypsin. Although relaxin also contains a number of basic residues including one pair of basic residues near the carboxyl terminus of the A-chain, the connecting peptide may be more exposed and hence may be more susceptible to tryptic degradation. ACKNOWLEDGMENTS We gratefully acknowledge Dr. Martin Rosenberg and Smith Kline & French Laboratories for the POTS expression vector and,% coli hosts; Dr. Billy Hudson for the use of his HPLC facilities; Dr. Gillian BryantGreenwood for the antiserum to porcine relaxin; Dr. Frank Simmen for a sample of porcine ovary used in the initial cDNA library construction; and Dr. Donald F. Steiner for providing us the oligonucleotide for the initial isolation of the porcine preprorelaxin cDNA. We also thank Dr. Audree Fowler, Paul Gardner, Douglas Larsen, and the star? in the Assay Services of Genentech for their excellent assistance in the amino-terminal sequence analysis, oligonucleotide synthesis, densitometric scanning, and bioassay of prorelaxin and relaxin, respectively. Protein sequencing and amino acid analysis performed at the UCLA Protein Microsequencing Facility was aided by BRS Shared Instrumentation Grant 1 SlORR05554-01 from the National Institutes of Health.
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RECOMBINANT 18. Devare, S. G., Shatzman, A., Robbins, Aaronson, S. A. (1984) Cell 36,43-49.
PORCINE K. C., Rosenberg,
PRORELAXIN M.,
and
19. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 20. Mott, Acad.
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21. Marston, (Glover, 22. Winkler,
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B. L., Berry,
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A. (1983)
132.365-375. 26. Burnette,
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coli
27. Fei, D. T. W., Gross, M. C., Lofgren, Chen, A. B. (1990) Biochem. Biophys.
J. L., Mora-Worms, M., and Res. Commun. 170,214-222.
28. Kwok, Endow.
G. D., and Niall,
S. C. M., Bryant-Greenwood, Res. Commun. 7,1-12.
H. D. (1980)
29. Stewart, A. G., Richards, H., Roberts, S., Warwick, J., Edwards, Bell, L., Smith, J., and Derbyshire, R. (1983) Nucleic Acids Res.
K.,
11,
Anal. B&hem.
CeU
30. Cabilly, S., Riggs, A. D., Pande, H., Shively, J. E., Holmes, W. E., Rey, M., Perry, L. J., Wetzel, R., and Heyneker, H. L. (1984) Proc. Natl. Acad. Sci. USA 81,3273-3277. 31. Walsh, 1260.
J. R., and Niall,
32. Charnley, W. A., Bagoyo, (1981) J. Endocrirwl. 88, 33. Bullesbach,
Anal.
IN
6597-6609.
F. A. 0. (1987) in DNA Cloning: A Practical Approach D. M., Ed.), Vol. 3, pp. 59-88, IRL Press, Oxford. M. E., and Blaber, M. (1986) Biochemistry 26,4041-4045.
23. O’Farrell, P. Z., Goodman, H. M., and O’Ferrell, 12,1133-1142. 24. Laemmli, U. K. (1970) Nature 227,680-685. 25. Anderson,
Proc. N&l.
EXPRESSED
112,195-203.
6004.
H. D. (1980)
Endocrinology
M. M., 89-96.
Bryant-Greenwood,
E. E., and Schwabe,
and
C. (1986)
Biochemistry
107, 1258G. D. 25,5998-
Vol.
OF BIOCHEMISTRY
294, No. 2, May
AND
BIOPHYSICS
1, pp. 579-585,
1992
Purification and Characterization of Recombinant Porcine Prorelaxin Expressed in Escherichia co/? G. Kesava Reddy,* Anthony B. Chen,t
Sripad Gunwar,* Carla B. Green,**2 and Simon C. M. Kwok*p3
David
T. W. Fei,?
*Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160; and tDepartment of Medicinal and Analytical Chemistry, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, California 94080
Received
October
24,1991,
and in revised
form
January
13,1992
In th$ report we describe the purification and characterization of recombinant porcine prorelaxin expressed in Eecherichia coli. Nucleotide sequence encoding porcine prorelaxin was inserted into an E. coli expression vector, POTS, and the recombinant plasmid was transformed into the E. coli host (AB120). Upon induction with nalidixic acid, the 19-kDa recombinant porcine prorelaxin was produced at a level of approximately 8% of the total accumulated cell protein. The recombinant prorelaxin was purified to homogeneity by CM-cellulose chromatography and reversed-phase HPLC, after refolding in the presence of reduced and oxidized glutathione and a low concentration of guanidine-HCl. The identity of the recombinant prorelaxin was confirmed by the correct size, immunoreactivity with antibodies against native porcine relaxin, and direct amino-terminal sequence analysis. Furthermore, the purified recombinant prorelaxin could be converted to the 6-kDa relaxin by limited digestion with trypsin. Trypsin was shown to cleave at the carboxyl side of Arg29 and Arg’37 residues of the recombinant prorelaxin, producing the des-A&‘-B29-relaxin, and degrade the 13-kDa connecting peptide into small peptides. Both the recombinant prorelaxin and converted relaxin were found to be biologically active in an in vitro bioassay for relaxin. Q 1992 Academic Press, Inc.
Relaxin is a 6-kDa peptide hormone which is composed of two peptide chains (A- and B-chains) linked by two interchain disulfide bonds. It is primarily synthesized by the corpus luteum during pregnancy, although a low level ‘This work was supported by U.S. Public Health Service HD24599. ’ Present address: Department of Anatomy and Cell Biology, versity of Kansas Medical Center, Kansas City, KS 66160. 3 To whom correspondence should be addressed. OOO3-9861/92 $3.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
Inc. reserved.
Grant Uni-
of relaxin activity has also been detected in the nonpregnant ovary, uterus, placenta, testes, and prostate (1, 2). The hormone exerts a profound effect on the reproductive tract during pregnancy. Its major biological functions are: (a) ripening of the uterine cervix, (b) relaxation of the pubic symphysis to facilitate birth, (c) inhibition of uterine contraction to prevent premature birth, and (d) development of the mammary gland. The amino acid sequences of relaxin have been determined by direct protein sequencing or deduced from the nucleotide sequences of the cDNAs for a number of species including pig (3-6), rat (7, 8), sand tiger shark (9), human (lo), spiny dogfish (ll), skate (121, whale (131, and monkey (14). Nucleotide sequence analysis of the relaxin cDNAs revealed that relaxin is synthesized as a single-chain 23-kDa preprorelaxin with the overall structure: signal peptide, B-chain, connecting peptide, and A-chain. During the biosynthesis of relaxin, the signal peptide is removed rapidly as the nascent peptide chain is translocated across the endoplasmic reticulum, producing the 19-kDa prorelaxin. Further processing of the prohormone into the 6-kDa relaxin is not well understood. In order to study the mechanism of conversion of prorelaxin to relaxin, a highly purified preparation of the prohormone is required. Although high molecular weight intermediates have been isolated from porcine ovaries (E), attempts to purify the prohormone from porcine ovaries have not been successful due to its extreme heterogeneity (unpublished result). We have, therefore, decided to produce porcine prorelaxin in Escherichia coli. We report herein the purification and characterization of the recombinant prorelaxin. MATERIALS
AND
METHODS
Bacterial strains and plasmids. E. coli strains (AR120, MM294cI+) and the E. coli expression vector (POTS) are lcmdly provided by Dr. Martin Rosenberg of the Smith Kline & French L&oratories. A plasmid, pPR 308-6, harboring a full-length porcine preprorelaxin cDNA was 579
580
REDDY
isolated from a porcine ovary cDNA library using a 5’-end-labeled oligonucleotide (21-mer) probe (kindly provided by Dr. Donald F. Steiner), which was synthesized based on the published nucleotide sequence of porcine preprorelaxin cDNA (6). The identity of the cDNA was confirmed by restriction endonuclease mapping and nucleotide sequence analysis using the chain termination method (16, 17). A &!&base-pair Construction of the prorekxin expression plasmid. RsaI/EcoRI fragment containing the coding sequence for promlaxm was isolated from plasmid pPR308-6 using a 5% polyacrylamide gel and was inserted into the expression vector, POTS (18), using standard recombinant DNA techniques (19), as depicted in Fig. 1A. The isolated fragment was blunt-ended with a Klenow fill-in reaction and ligated to a synthetic BamHI linker (8-mer). The BamHI-linkered fragment was then subcloned into the BamHI site of the POTS expression vector. The resulting recombinant plasmid was first transformed into E. coli MM294cP. Minipreparations of DNA were prepared as described by Del Sal et ol. (17) for confirmation of orientation by XhoI/BglII digestion and by nucleotide sequence analysis using a sequence-specific primer (16,17). Finally, the recombinant plasmid was transformed into E. coli AR120 for nalidixic acid induction. The clone was designated pPR754AR120. Growth and induction of bacteria Growth and induction of pPR754AR120 was carried out essentially as described by Mott et al. (20). Cultures were grown in maximal induction medium [3.2% tryptone, 2% yeast extract, 1X M9 salts, 0.1 mM MgSO,, 0.001 mM FeCl,, and 100 &g/ml ampicillin (20)] at 37’C until Am reached 1.0 and were then induced with nalidixic acid at a final concentration of 40 fig/ml for 16 h at 37’C. The 16-h induction was chosen as a matter of convenience, since the expression reached a maximum after 5 h of induction and the expressed protein is stable even after 21 h of induction (20). Isolation andpurification of recombinuntproreluxin. The E. coli cells grown and induced as described above were harvested by centrifugation and lysed with lysozyme and deoxycholic acid, and the inclusion bodies were washed with buffer containing 0.5% Triton X-100 and 10 mM EDTA’ as described by Marston (21). Subsequent steps for the isolation of the recombinant prorelaxm were adapted from the procedure described by Winkler and Blaber (22). The washed inclusion bodies were purifled by centrifugation through a 50% glycerol cushion and then solubilixed by 6 M guanidine-HCl in 50 mM Tris-HCl (pH 8.0) containing 100 mM NaCl and 1 mM EDTA (1 ml/g cells) at 4°C for 16 h. Insoluble material was removed by centrifugation. The supernatant was initially diluted with 5 vol of 50 mu Tris-HCl (pH 9.0) containing 5 mM EDTA and then diluted further to Auu, < 1.0 with 1 M guanidine-HCI in the same buffer. The extracted proteins were allowed to refold at 4’C for 24 h in the presence of 0.2 M arginine, 0.005% Tween 80, 1.25 mu reduced glutathione, and 0.25 mM oxidized glutathione (22). The refolded proteins were then dialysed against 10 mu ammonium acetate (pH 5.5) containing 1 mM EDTA and 20 pg/ml PMSF, and insoluble material was removed by centrifugation. The clarified protein sample was passed through a 2.5 X 20-cm column of CM-cell~ose (CM-52, Whatman) preequilibrated with 10 mM ammonium acetate (pH 5.5) containing 1 mM EDTA and 20 pg/ml PMSF (starting buffer), and unabsorbed proteins were washed with the same buffer. The absorbed proteins were eluted with a linear gradient of O0.2 M NaCl formed by 300 ml of starting buffer and 300 ml of 0.2 M NaCl in starting buffer. Fractions of 2.5 ml were collected at a flow rate of 15 ml/h. Aliquots of the fractions were analyzed on a 15% SDSpolyacrylamide gel (24), followed by Western blot analysis (described below). Prorelaxin containing fractions were pooled, dialyzed against 10 mM ammonium acetate (pH 5.5) containing 1 mM EDTA and 20 pg/ ml PMSF, and then lyophilized.
’ Abbreviations used: EDTA, ethylenediaminetetraaceta~, PMSF, phenylmethylsulfonyl fluoride; HPLC, high-performance liquid chromatography; SDS, sodium dodecyl sulfate; DFP, diisopropyl fluorophosphate; TPCK, r.,-l-p-tosylamino-2-phenylethyl chloromethyl ketone.
ET
AL.
Subsequent purification of recombinant prorelaxin was carried out by reversed-phase HPLC using a Vydac Cn column. The column was equilibrated with 30% acetonitrile and 0.1% tritluoroacetic acid. Approximately 2 mg of partially purified recombinant prorelaxin from the CM-cellulose column was dissolved in 1 ml of 50 mu Tris-HCI (pH 7.5), adjusted to 0.5% trifluoroacetic acid, and injected into the column. A 30-min gradient of 30-70% acetonitrile was used to elute the proteins at a flow rate of 2 ml/min. Fractions were collected manually for various
A l?zoRI
I!coRI
1
;~~l~+ Eco RI digestion
I
I
Klemw fill-in Barn HI linkers Barn tll dtgestion
Barn HI digestion Dcphnsphmylation
BlUtipd
1
Ligation
B&I HI
B
:zoFrtion
~tor.ldn I
Met
Thr Asn Asp ACG AAC CAT...
AT-
Asp
Pro
*
a&mar linker
FIG. 1. (A) Construction of the prorelaxin expression plasmid pPR754-AR120. The details of the construction are described under Materials and Methods. The open box represents the coding sequence of prorelaxin with the hatched region indicating the coding sequence for the prepeptide. The closed box represents the sequence derived from phage X. 9JPi,, bacteriophage lambda operator and promoter; cI1 r.b.s., cl1 ribosome binding site; to,lambda phage terminator; Amp’, ampicillin resistance. (B) Coding sequence present in pPR754-AR120 at the junction between the prorelaxin and the vector. The initiation codon is provided by the POTS expression vector. The codons for the second and third residues (Asp-Pro) are contributed by the linker (shown in italics). The remaining coding sequence is derived from the porcine prorelaxin cDNA.
RECOMBINANT
PORCINE
peaks and lyophilized immediately. The peak corresponding binant prorelaxin was identified by SDS-polyacrylamide phoresis and Western blot analysis.
PRORELAXIN to recomgel electro-
Limited digestion of recombinant prorelaxin with trypsin. TPCKtrypsin (Sigma) was dissolved in 0.001 N HCl at a concentration of 1 mg/ml. Purified recombinant prorelaxin was dissolved in water at a concentration of 5 pg/Fl. Aliquots of 5 pg of recombinant pmrelaxin were pipetted into a series of microcentrifuge tubes containing 20 /.d of 50 mM Tris-HCl (pH 8.1) and 11.5 mM CaClx. TPCK-trypsin was then added at different enzyme-to-substrate ratios (1:lOO to 1:500 w/w), and the reaction mixture was vortexed briefly and incubated for 5 min at 37’C. The trypsin activity was arrested by freezing on dry ice and the samples were dried in a vacuum evaporator. The tryptic peptides were analyzed by urea-SDS-polyacrylamide gel electrophoresis (25). To isolate the converted relaxin from limited tryptic digest of recombinant pmrelaxin, the digested material was acidified with trifluomacetic acid (to a final concentration of 0.5%) and injected into the Vydac Cis column equilibrated with 10% acetonitrile. A 50-min gradient of lo50% acetonitrile was used to elute the converted relaxin at a flow rate of 1 ml/min. Fractions were collected manually for each peak and lyophilized immediateiy. The converted relaxin peak was identified by urea-SDS-polyacrylamide gel electmphoresis and Western blot analysis. Western blot analysis. The procedure of Bumette (26) was used with minor modifications. The separated proteins were electrophoretically transferred to nitrocellulose, blocked with 5% nonfat dry milk, and reacted with antiserum against porcine relaxin (kindly provided by Dr. Gillian Bryant-Greenwood). After thorough washing, the blot was incubated with a mixture of alkaline phosphatase-conjugated anti-rabbit IgG (Sigma) and streptavidin-alkaline phosphatase conjugate (Pierce). The pmrelaxin/relaxin bands and biotinylated molecular weight markers were then visualized with chromogenic substrates (nitro blue tetrazonium and 5-bromo-4-chloro-3-indolyl phosphate). Carboxypeptidase digestion. The HPLC-purified converted relaxin (20 pg) was digested with DFP-treated carboxypeptidase A or B (Sigma) as described by Schwabe et al. (13). The amino acid(s) released was determined by amino acid analysis. A reagent blank consisting of buffer and enzyme but no relaxin was used to subtract the background.
EXPRESSED
IN
Escherichia
581
coli
residues (Gln-Ser) at the amino terminus replaced by Met-Asp-Pro residues (Fig. 1B). The Met codon was provided by the expression vector, and the Asp-Pro codons were furnished by the BamHI linkers. Purification and characterization of recombinant prorelaxin. Although the recombinant prorelaxin represents 8% of the total E. coli proteins, we took advantage of the fact that prorelaxin forms aggregates known as inclusion bodies. The bulk of bacterial proteins were removed by purifying the inclusion bodies before the solubilization of the recombinant prorelaxin. Other bacterial proteins were removed as precipitates when the refolded proteins were dialyzed against buffer containing no denaturing agents. Further removal of bacterial proteins was accomplished with CM-cellulose chromatography, which also served to concentrate the recombinant prorelaxin from a large volume of diluted protein sample (2.1 liters). Only one major absorbed protein peak was eluted from the CM-cellulose column (data not shown). Western blot analysis showed that this peak contained the recombinant prorelaxin (data not shown). Further purification by reversed-phase HPLC revealed that this partially purified recombinant prorelaxin was composed of three major peaks (designated A, B, and C in Fig. 2). Western blot analysis showed that only peak B, which was eluted at 41% acetonitrile, contained the recombinant prorelaxin (data not shown). The yield of prorelaxin from a 3-liter culture was 2.4-3.0 mg. The results from SDS-polyacrylamide gel electrophoresis suggested that recombinant prorelaxin was purified to homogeneity by two chromatographic steps (Fig. 3, lane
Determination of amino-terminal sequence and amino acid composition. The amino-terminal sequence and amino acid composition analyses of purified recombinant prorelaxin and converted relaxin were performed by Dr. Audree Fowler at the University of California, Protein Microsequencing Facility, Los Angeles, California.
r
Bioassay. The biological activities of recombinant prorelaxin and converted relaxin were determined with the in vitro bioassay using human endometrial cells as described by Fei et al. (27). The concentration of recombinant prorelaxin and converted relaxin were determined spectrophotometrically using the values of Alx,, of 20.5 and 2.0, respectively. The AIZm, value of prorelaxin was determined by amino acid analysis of an aliquot of a sample of known absorbance at 280 nm and that of relaxin was reported earlier (28).
RESULTS
Corzstruction of the prorehxin expression plasmid. A plasmid harboring a full-length porcine preprorelaxin cDNA was used as starting material for the construction of the prorelaxin expression plasmid. In order to express porcine prorelaxin in E. coli, the coding sequence for the prepeptide was removed by RsaI digestion, which also removed the first two codons of the B-chain. The truncated prorelaxin cDNA was then subcloned into the BamHI site of the POTS expression vector using BarnHI linkers (Fig. 1A). This expression plasmid thus produced a variant form of porcine prorelaxin with its first two “authentic”
: I
(
I
I
38 40 42 %ACETONITRILE FIG.
I
I
44
2. Purification of recombinant prorelaxin by reversed-phase HPLC. Partially purified sample from the CM-cellulose column (2 mg) was applied onto the Vydac Cis column and eluted with a linear gradient of acetonitrile as described under Materials and Methods. Three major peaks were obtained in the elution profile. Peak B, which was eluted at 41% acetonitrile, contained recombinant prorelaxin as confirmed by Western blot and amino-terminal sequence analyses.
582
REDDY MW
1
2
3
4
5
6
FIG. 3. Analysis of the products released by limited trypsin digestion of recombinant prorelaxin on urea-SDS-polyacrylamide gel. About 5 pg of recombinant prorelaxin was digested with various concentrations of TPCK-trypsin at 37°C for 5 min, lyophilized, boiled in sample buffer containing no P-mercaptoethanol, and analyzed on a urea-SDS-polyacrylamide gel. The enzyme to substrate ratios (w/w) are as follows: lane 1, 1:lOO; lane 2, 1:200, lane 3, 1:300; lane 4, 1:400; lane 5, 1:500; and lane 6, without enzyme. The molecular weights of 19 and 6 kDa marked the migration of intact recombinant prorelaxin and insulin, respectively, which were also electrophoresed in the absence of &mercaptoethanol.
6). Amino-terminal sequence analysis of the purified recombinant prorelaxin yielded the following sequence: Met -Asp -Pro -Thr -Asn- Asp- Phe- Ile- Lys- Ala -CysGly-Arg-Glu-Leu-Val-Arg-Leu-Trp. This sequence agreed perfectly with that expected from vector construction and the published sequence deduced from porcine preprorelaxin cDNA (6). The first two residues (Gln-Ser) in the natural prorelaxin were replaced by Met-Asp-Pro at the amino-terminal end of recombinant prorelaxin. The amino-terminal Met residue of recombinant prorelaxin is neither blocked by a formyl group nor removed. Limited tryptic digestion. The results from limited tryptic digestion studies indicated that the recombinant prorelaxin could be converted by trypsin to a polypeptide of the size of relaxin. At low concentrations of trypsin, recombinant prorelaxin was partially cleaved, giving rise to a band with slower mobility than the intact prohormone (Fig. 3, lanes 4 and 5). The conversion of recombinant prorelaxin to relaxin occurred gradually at a 1:300 (w/w) enzyme-to-substrate ratio, as visualized by the appearance of a band corresponding to the 6-kDa relaxin on ureaSDS-polyacrylamide gel electrophoresis (Fig. 3, lane 3). At a 1:lOO (w/w) enzyme-to-substrate ratio, only the conversion product of the size of relaxin can be observed during the 5-min incubation period at 37°C (Fig. 3, lane 1). When this tryptic digest was chromatographed on the Vydac Cis column, one major peak was eluted at 39% acetonitrile (Fig. 4). Amino-terminal sequence analysis of the purified conversion product revealed two residues per cycle in approximately equimolar amounts. The results, when compared with the sequence of the B-chain (stated above), yielded the following sequences: Met-Asp-
ET
AL.
Pro-Thr-Asn-Asp-Phe-Ile-Lys-Ala for the B-chain and Met-Thr-Leu-Ser-Glu-Lys-Cys-Cys-Gln-Val for the A-chain. The latter agrees perfectly with the amino-terminal sequence of the A-chain of the native relaxin (3, 4), except that the Ar$l residue in the native relaxin is missing in the converted relaxin. Amino acid analysis of the HPLC-purified, converted relaxin after digestion with carboxypeptidase B revealed the release of 186 pmol of arginine. However, a similar experiment with carboxypeptidase A failed to produce any significant amount of released amino acids above background. Both the recombinant prorelaxin and the converted relaxin were found to be biologically active in an in vitro bioassay for relaxin, with 1 maximal activities (ED& of 11.7 and 6.2 nM, respectively (Fig. 5). During these studies we also examined the stability of the converted relaxin and the fate of the connecting peptide by the action of trypsin following a lo-, 20-, and 30min incubation period with the enzyme-to-substrate ratio kept constant (1:lOO). The results indicated that the relaxin band was observed in samples incubated at lo- and 20-min intervals. However, no relaxin band was detected on the urea-SDS-polyacrylamide gel if the incubation period was over 30 min (data not shown). On the other hand, the expected 13-kDa connecting peptide was not observed even after 5 min of incubation. -
12
20
26
36
44
% ACETONITRILE
FIG. 4. Separation of converted relaxin from limited tryptic digest of recombinant prorelaxin by reversed-phase HPLC. Limited tryptic digest of recombinant prorelaxin (1 mg) was applied onto the Vydac C18 column and eluted with an acetonitrile gradient as described under Materials and Methods. The single major protein peak, which was eluted at 39% acetonitrile, was found to be converted relaxin by Western blot and amino-terminal sequence analyses. The arrow designates position where recombinant prorelaxin (PR) elutes.
RECOMBINANT
PORCINE
100 Porcine
PRORELAXIN
10' Proreloxin
102 CnMI
-f-
L
10-l
I
I
100
I
10' Porcine
Reloxin
102 Cd.4
1
FIG. 5. In vitro bioassay of recombinant prorelaxin and trypsin-convetid relaxin. Human uterine endometrial cells were treated with various dilutions of recombinant prorelaxin (top panel) or converted relaxin (bottom panel), and the amounts of intracellular CAMP produced were determined by radioimmunoassay. Each value is the mean f SD of triplicate determinations. Since maximal CAMP response varies in different batches of cells, the biological activities were expressed as $ maximal activities (ED,), which were found to be 11.7 and 6.2 nM for recombinant prorelaxin and converted relaxin, respectively.
DISCUSSION In the present paper we have described the purification and characterization of recombinant prorelaxin produced by E. coli. The identity of prorelaxin was confirmed by the correct size, immunoreactivity with the antibodies against native porcine relaxin, direct amino-terminal sequence analysis, and biological activity in an in vitro
EXPRESSED
IN
Escherichia
coli
583
bioassay. Furthermore, we have also demonstrated the conversion of the recombinant prorelaxin to relaxin by a limited digestion with TPCK-trypsin. Expression of porcine prorelaxin in E. coli has also been reported by another group of investigators (29). However, to the best of OUI knowledge, no report has been made on the purification and characterization of the recombinant prorelaxin. During the purification of recombinant prorelaxin, we have attempted several procedures. The procedure described in this paper was an adaptation of the method of Winkler and Blaber (22) and was used with success. The procedure involves extraction of the recombinant prorelaxin with 6 M guanidine-HCl, followed by refolding of the crude extract in the presence of oxidized and reduced glutathione and 1 M guanidine-HCl. By refolding the recombinant prorelaxin in the presence of oxidized and reduced glutathione prior to chromatography, the use of denaturing agents can be eliminated in the subsequent purification procedures. Furthermore, the recombinant prorelaxin can be purified to homogeneity by merely two chromatographic steps. In the other procedure, the recombinant prorelaxin was converted into an S-sulfonate derivative by the reaction with sodium sulfite and sodium tetrathionate as described by Cabilly et al. (30). The Ssulfonated recombinant prorelaxin was then subjected to gel filtration and ion-exchange chromatography using buffers containing denaturing agents. This procedure proved to be unsuccessful for the purification of recombinant prorelaxin. The immunoreactive materials appeared as a smear in the chromatographic profiles (data not shown). In this report we use TPCK-trypsin as a model enzyme to study the conversion of recombinant porcine prorelaxin and to establish a “conversion assay” for the prorelaxin processing protease( Although trypsin is not the authentic prorelaxin processing protease, it has been used successfully to convert high molecular weight forms of porcine relaxin to the 6-kDa relaxin (15). Trypsin can easily cleave at the carboxyl side of the cluster of four basic residues (residues 131-134) near the connecting peptide/A-chain junction (6). On the other hand, although the processing enzyme for the B-chain/connecting peptide junction is not a trypsin-like enzyme, trypsin may cleave at the carboxyl side of Arg2’, which is located only three residues away on the amino side of the proposed cleavage site (6). Our results on limited digestion of recombinant prorelaxin with trypsin showed that it can be converted to the 6-kDa relaxin. Amino-terminal sequence analysis of the converted relaxin reveals that the Arg residue at the amino terminus of the A-chain is removed. This suggests that prorelaxin is cleaved at the carboxyl side of Arg137 instead of the cluster of four basic residues (positions 131-134) near the connecting peptide/A-chain junction (6). On the other hand, cleavage at the B-chain/ connecting peptide junction was inferred indirectly from the experiments with carboxypeptidases A and B. Since
584
REDDY
the carboxyl-terminal Cys residue of the A-chain is involved in the formation of a disulfide bond, carboxypeptidases A and B will only release amino acids from the carboxyl terminus of the B-chain. Carboxypeptidase B digestion of the converted relaxin released only arginine, while carboxypeptidase A did not release any amino acids. The results are consistent with the prediction that the cleavage may occur between Arp and Th$O, leaving Arp as the carboxyl terminus of the B-chain. The converted relaxin is thus des-Are’-B29-relaxin. Both the recombinant prorelaxin and converted relaxin were found to be biologically active in the in vitro bioassay for relaxin. However, whether the biological activity of the recombinant prorelaxin is due to its intrinsic property or on-site conversion on the surface of target cells remains to be determined. Conversion with trypsin increased the biological activity of relaxin by merely twofold. Although earlier observations have shown that high molecular weight forms of relaxin are biologically active (15), the present study unequivocally demonstrates for the first time that prorelaxin is biologically active. On the other hand, the biological activity of the converted relaxin is about threefold less than that of the native porcine relaxin, which has a EDso value of 1.98 nM (27). The discrepancy in the biological activities of the recombinant and native relaxins may be explained by several structural differences. First, the recombinant relaxin differs slightly from the native relaxin in that it contains Met-Asp-Pro residues instead of Gln-Ser residues at the amino terminus of the B-chain as a result of the expression plasmid construction. Second, not all the recombinant relaxin molecules may be refolded correctly. Third, the removal of Arg at the amino terminus of the A-chain and the Thr30-Ala31-Leu32 residues at the carboxyl terminus of the B-chain may lead to the lower biological activity of the converted relaxin in the in vitro bioassay. However, it is interesting to note that the removal of Thr3’-Ala31Leu32 at the carboxyl terminus of the B-chain will generate a molecule corresponding to the B29 relaxin (CM-a’) which possesses full biological activities in the in vivo bioassay (31,32) and that the removal of the Arg residue at the amino terminus of the A-chain has little effect on the biological activity of the relaxin, also in the in vivo bioassay (33). We also looked at the fate of the connecting peptide, as it is better conserved than the A- and B-chains in relaxins of different species studied thus far and hence may possess a biological function(s) other than folding the two chains into proper conformation. In our experiments, we found that at a 1:lOO (w/w) enzyme-to-substrate ratio, recombinant prorelaxin is optimally converted to relaxin during a 5-min incubation period at 37°C. The converted relaxin is stable under these conditions for up to 20 min of incubation, but is completely degraded during longer incubation. However, even after 5 min of incubation, we are unable to detect the expected 13-kDa connecting pep-
Fe.1 --
AL. AT
tide on the SDS-polyacrylamide gel. A closer examination on the amino acid sequence of the porcine prorelaxin connecting peptide (6) reveals at least 16 possible cleavage sites for trypsin. Although relaxin also contains a number of basic residues including one pair of basic residues near the carboxyl terminus of the A-chain, the connecting peptide may be more exposed and hence may be more susceptible to tryptic degradation. ACKNOWLEDGMENTS We gratefully acknowledge Dr. Martin Rosenberg and Smith Kline & French Laboratories for the POTS expression vector and,% coli hosts; Dr. Billy Hudson for the use of his HPLC facilities; Dr. Gillian BryantGreenwood for the antiserum to porcine relaxin; Dr. Frank Simmen for a sample of porcine ovary used in the initial cDNA library construction; and Dr. Donald F. Steiner for providing us the oligonucleotide for the initial isolation of the porcine preprorelaxin cDNA. We also thank Dr. Audree Fowler, Paul Gardner, Douglas Larsen, and the star? in the Assay Services of Genentech for their excellent assistance in the amino-terminal sequence analysis, oligonucleotide synthesis, densitometric scanning, and bioassay of prorelaxin and relaxin, respectively. Protein sequencing and amino acid analysis performed at the UCLA Protein Microsequencing Facility was aided by BRS Shared Instrumentation Grant 1 SlORR05554-01 from the National Institutes of Health.
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G. D. (1982)
Endocr.
Rev. 3,62-90.
2. Sherwood, 0. D. (1988) in The Physiology of Reproduction (Knobil, E., and Neill, J., Eds.), pp. 585-673, Raven Press, New York. 3. James, R., Niall, H., Kwok, S., and Bryant-Greenwood, G. (1977) Nature 267,544-546. 4. Schwabe, C., McDonald, J. K., and Steinetz, B. G. (1976) Bicchem. Biophys. Res. Commun. 70,397-405. 5. Schwabe, C., McDonald, J. K., and Steinetz, B. G. (1977) Biochem. Biophys. Res. Commun. 75,503-510. 6. Haley, J., Hudson, P., Scanlon, D., John, M., Cronk, Tregear, G., and Niall, H. (1982) DNA 1.155-162. 7. John, M. J., Borjesson, B. W., Walsh, Endocrinology 108, 726-729. 8. Hudson, P., Haley, J., Cronk, Nature 291, 127-131.
M.,
M., Shine,
J. R., and Niall, Shine,
J., and Niall,
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