hr. J. Biochem. Vol. 23, No. 1, pp. 107-114, Printed in Great Britain. All rights reserved
0020-711X/91
1991
$3.00 + 0.00
Copyright 0 1991Pergamon Press plc
EXPRESSION AND PARTIAL PURIFICATION OF HUMAN PROLACTIN IN ESCHERICHIA COLI M. S. GILBERT, P. J. LOWRY, M. G. CASTRO, R. J. WDDDSand D. SAVVA* School of Animal and Microbial Sciences, Department of Biochemistry & Physiology, University of Reading, Whiteknights, P.O. Box 228, Reading RG6 2AJ, England [Tel. (0734) 875123; Fax (0734) 310180] (Received 3 May 1990)
Abstract-l. Human prolactin has been expressed in Escherichia co/i. A cDNA fragment coding for the signal sequence and the full length prolactin molecule was cloned into the expression vector pUR291 which directs the synthesis of a B-galactosidase prolactin fusion protein when expressed in E. cofi. 2. Cultures of E. coli harbouring the recombinant plasmid pJMBG62 produced a fusion protein of the appropriate molecular weight which was detected by Western blot analysis using a polyclonal antibody raised against pituitary-derived human prolactin. 3. The fusion protein was isolated from inclusion bodies in a partially pure form and it was used as immunogen to raise antibodies against human prolactin. 4. When this partially purified fusion protein was injected into rabbits it generated antisera with good prolactin titres in animals which were rested for one year following a disappointing primary immunization with purified human prolactin.
INTRODUCTION Human prolactin is a single chain polypeptide of 199 amino acids and a molecular weight of 23,000. It is synthesized and released from the lactotrophic cells of the anterior pituitary and belongs to a set of hormones collectively known as the ‘Prl Set’ (Cooke et al., 1981) which includes growth hormone and chorionic somatomammotropin (placental lectogen). It is believed that the ‘Prl Set’ hormones evolved from a common ancestral gene by subsequent sequence divergence (Catt et al., 1967), as indicated by similarities in their amino acid sequences and by overlaps in their biological and immunological properties (Niall et al., 1973). In humans the major area of action of prolactin is on the mammary gland where it is involved in the stimulation of postpartum lactation. It is also involved in other aspects of pregnancy and has some physiological effects in men (Moltich, 1985; Blackwell, 1985). Several disorders have been associated with prolactin. In women, abnormal levels of serum prolactin have been linked with such conditions as galactorrhea, anovulation with amenorrhea, hypooestrogenism and hyperprolactinaemia which has been shown to be common in some 10% of all female infertilities. In men, hyperprolactinaemia has been linked to sexual disorders such as impotence and ejaculatory incompetence (Nickel, 1984). The ability to detect circulating levels of prolactin in patients with the above disorders would be a useful diagnostic tool. The main methods currently employed for the detection of circulating prolactin are radioimmunoassays (Sinha et al., 1973) and immunoradiometric assays (Hodgkinson et al., 1984). Both of these depend on the availability of high specificity *To whom all correspondence Bc2311-P
should be addressed
antiserum to prolactin and these in turn depend on the availability of the human hormone for use as an antigen but its immunogenicity can sometimes be poor. Recent advances in recombinant DNA technology provide a means of producing human prolactin fusion proteins in bacteria such as Escherichiu coli. Also, a molecular biology approach would eliminate the need of handling human tissues and will provide a non-infectious source of prolactin, eliminating contact with viruses such as HIV and hepatitis among others. This paper describes: (1) the expression of human prolactin in E. coli using a pUR expression vector (Ruther and Muller-Hill, 1983); (2) the partial purification of a /I-galactosidase fusion protein containing the full length human prolactin at its carboxy-terminus; and (3) the use of this fusion protein to raise antibodies against human prolactin. MATERIALSAND METHODS Piasmidr and bacterial strains
Human prolactin cDNA cloned into plasmid pBR322 [a generous gift from Dr J. Martial, Institute of Chemistry, Sart Tilman, Belgium; Cooke et al. (1981)] was maintained in E. coli HBlOl (Boyer, 1969). The expression vector pUR291 was supplied by Dr U. Ruther (University of Kiiln, West Germany) and it was maintained in E. co/i BMH 71-18 (Messing et al., 1977). Bacterial strains were transformed using the CaCl, procedure of Mandel and Higa (1970), and transformants were selected on LB (10 g/l Bacto-tryptone. 5 g/l Bacto-yeast extract, 5 g/l NaCl, i g/i’glucose; bacterialogical powders were from Lab M Ltd. Salford. Manchester. U.K.) agar plates containing ampicilhn (50&ml). Large scale plasmid preparations were performed using the method described by Bimboim and Doly (1979) with the modifications described by Sawa (1983). 107
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M. S. GILBERT~~ al.
Construction and identification of recombinant plasmids
Restriction endonucleases and T4 DNA ligase were obtained form Bethesda Research Laboratories (BRL, Paisley, Strathclyde, U.K.) and were used as recommended by the supplier. b-galactosidase was supplied by Sigma Chemical Company Ltd. (Poole, Dorset, U.K.). DNA restriction fragments were separated on 0.8% (w/v) agarose gels in TBE (89 mM Tris base, 90 mM boric acid, 1 mM Na, EDTA, pH 8.0). When necessary, fragments were purified from low melting point agarose gels (Weislander, 1979). The recombinant pBR322 plasmid containing the human prolactin cDNA [Fig. l(a)] was cleaved with PstI to generate three DNA fragments: the pBR322 vector and two smaller fragments of 588 and 367 bp. The 588 bp fragment codes for 10 glycines, an asparagine, the 28 amino acids of the signal sequence and the first 157 amino acids of human prolactin; this was ligated with PsrI-cleaved expression vector pUR291 to generate plasmid pJMBG60 [Fig. l(b)]. This plasmid was used to construct an expression plasmid containing the complete cDNA for human prolactin. Briefly, after digestion with BglII and Hind111 the 70 bp fragment was removed and replaced with the 222 bp BglII-Hind111 fragment from the original prolactin cDNA [Fig. l(a)]. The ligation mixture was used to transform E. co/i BMH 71-18. Plasmid DNA was isolated from some of the transformants and analysed with restriction enzymes. Recombinant plasmids were identified by in situ colony hybridization (Grunstein and Hogness, 1975). Bacterial colonies were transferred from the agar plates onto
Hybond-N membranes as described by the supplier of the membrane (Amersham International plc, Amersham, Bucks., U.K.). Transformants were screened using the 700 bp AoaII-AuaII fragment of the prolactin cDNA [Fig. l(a)] which was labelled to a sp. act. of 2 x lo9 dpm/pg DNA using [a-32P]dCTP (3000 Ci/mmol; Amersham International) and the method of Vogelstein and Feinberg (1983). Hybridization was performed at 65°C overnight in 6 x SSC (0.9 M NaCl, 0.09 M trisodium citrate, pH 7.0) containing 0.5% (w/v) dried skimmed milk. Membranes were washed at 65°C using 0.1 x SSC (15 mM NaCl, 1.5 mM trisodium citrate, pH 7.0) containing 0.1% (w/v) sodium dodecyl sulphate (SDS) and were autoradiographed. Analysis of recombinant proteins by SDS-polyacrylamiak electrophoresis and Western blotting
Cultures harbouring recombinant plasmids were grown and processed for SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting as described by Grewal et al. (1989). Protein concentrations were determined by a Coomassie Blue assay using Pierce Reagent (Pierce Chemical Co., Rockford, Ill., U.S.A.). The activity of b-galactosidase was determined as described previously (Pardee et al.. 1959). The primary antibody used for the Western blots was a rabbit anti-human prolactin antibody (P2) and the second antibody was a peroxidase-conjugated goat anti-rabbit IgG Fc fragment specific antibody (Jackson Immunoresearch Laboratories Inc., Pa, U.S.A.). Antibody complexes were detected by the appearance of coloured bands after the membranes were incubated in 100 ml PBS (10 mM sodium
(a)
-28
PSJ
gel
+I
4!Ld
100 bp
(b)
Fig. 1. (a) Partial restriction map of the human prolactin cDNA. The solid, open and hatched boxes represent the signal sequence, the coding sequence and the 3’ untranslated cDNA respectively. The solid lines indicate homopolymer tails. Numbers refer to amino acid residues. (b) Schematic diagram and partial restriction map of plasmids pJMBG60 and pJMBG62 containing different portions of human prolactin cDNA (not drawn to scale). For the cDNA closed, open and hatched boxes as in Fig. l(a). The /I-galactosidase (la&) and the ampicillin resistance (ApR) genes on the plasmid are also shown.
Human prolactin in E. coli phospahte buffer pH 7.5, 0.13 M sodium chloride) containing 20mg of 3,3-diaminobenzidine (Sigma Chemical Co., Poole, Dorset, U.K.) and 10~1 H,O, (100~01). All incubations were carried out at room temperature with gentle shaking. Isolation of inclusion bodies
Inclusion bodies produced by cultures containing plasmid pJMBG62 were isolated according to Marston (1985); in order to ensure complete lysis of the cells, an additional sonication step was introduced (three 30 set bursts). The cell lysate was centrifuged at 10,OOOgfor 15 min, the supernatant was removed and the pellet containing the inclusion bodies was resuspended in 0.04~01 lysis buffer (50mM Tris-HCl pH 8.0, 1 mM Na, EDTA pH 8.0, 0.1 M NaCl) containing 0.5% (v/v) Triton X-100. After incubation for 30 min at room temperature, the sample was centrifuged as before and the pellets washed in 0.1 ~010.1 M Tris-HCl pH 8.0 containing different amounts of urea (0.1 M-5.0 M) in order to determine the highest concentration of urea at which the fusion protein was still insoluble but which would remove as many E. coli contaminating proteins as possible. Aliquots from each stage were analysed by electrophoresis on SDSpolyacrylamide gels. Purification, solubilization and renaturation of fusion protein
Cell pellets containing inclusion bodies were first washed with lysis buffer containing 0.5% (v/v) Triton X-100 and then with 0.1 M Tris-HCl-pH 8.0.containing 2.5 M urea before solubilization in 0.1 vol 0.1 M Tris-HCl DH 8.0 containing 8 M urea; samples were incubated at room temperature for 20 min and centrifuged at 10,OOOg for 30 min. The urea was removed from the samples by means of serial dilutions in 50 mM Tris-HCl pH 8.0, 1mM Na, EDTA pH 8.0, 100mM NaCl, 0.5% (v/v) 2-mercaptoethanol, 20% (v/v) glycerol as follows: an equal volume of dilution buffer was added dropwise to the samples and mixed gently for 3 hr at 4°C to reduce the urea concentration to 2 M before dialysis of the samples for 24 hr at 4°C against 50 mM Tris-HCl pH 8.0, 1 mM Na, EDTA, 0.5% (v/v) 2-mercaptoethanol, 20% (v/v) glycerol. Radioimmunoassay for human prolactin The two-antibody method described by Beck et al. (1965) was used. The total volume of 250 ~1 consisted of 100 ~1 of a l/500 dilution of the prolactin antibody (P2), 50~1 1251-labelled nrolactin (ca 15.000 cnm: Netria. 36 uCi/u e protein) and 100 ~1 of prolactin Astandards ‘(Netria,’ ‘ST Bartholomew’s Hospital, London), ranging in concentration from 800 to 0.78 ng/ml, or dilutions of the samples to be tested. All dilutions were made up in 50 mM sodium phosphate buffer pH 7.4 containing 0.5% (w/v) bovine serum albumin. Samples were incubated for 24 hr at room temperature before precipitation (30 min at room temperature) with 200 ~1 of precipitating solution (50 mM sodium phosphate buffer pH 7.4, 10% v/v sheep anti-rabbit Fc antiserum, 4% w/v polyethylene glycol6000, 1% v/v normal rabbit serum; this solution was incubated at 4°C for 24 hr prior to use). The sheep anti-rabbit Fc antiserum was obtained from Polvclonal Antibodies Ltd (Dvfed. Wales). After addition of -2 ml 0.15 M NaCl, samples were centrifuged at 2500 g for 20 min at 4”C, the supematants were removed and the radioactivity in the pellets was determined on a LKB gamma counter. Immunization of rabbits
A total of 10 rabbits (MGl-MGlO, New Zealand white, 2 kg in weight; Regal Rabbits, Great Bookham, Surrey, U.K.) were employed for this experiment. Two of the rabbits (MGI and MG2) had previously been iniected over a period of a year with purified natural human prolactin (Hodgkinson et al., 1984) and had been rested for 1 year.
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All rabbits were primed by intramuscular injections at three sites with a mixture consisting of 3 ml Freund’s complete adjuvant (except in the case of rabbits MGI and MG2 where the mixture contained Freund’s incomplete adjuvant), 5~1 Brij’s solution (30% v/v; Sigma) and 1 ml saline containing the required amount of the immunogen. The immunogen used was 2.3 mg partiallypure prolactin fusion protein (equivalent to 1OOpg pure nrolactin) for rabbits MGl-6 and 50 tirr of purified human prolactin’ for rabbits MG7-10. Subsequent injections (at monthly intervals) were similar except that Freund’s incomplete adjuvant was used and the amount of immunogen was halved. Bleeds (20 ml) were taken from the ear of each rabbit 10 days after each injection and the clots were allowed to form an contract at room temperature for 24 hr. The sera were removed and subsequently stored at -20°C. Prolactin and /I-galactosidase antibody titres were assayed by measuring the amount of 12SI-labelled prolactin and 1251-labelled/Igalactosidase bound by a series of semm dilutions ranging from l/250 to l/64,000. Each assay had a total volume of 200~1, consisting of 100~1 antibody (diluted in 50mM sodium phosphate buffer pH 7.4, 0.5% w/v bovine serum albumin) and 100 ~1 of 50mM sodium phosphate buffer pH 7.4, 0.5% (w/v) BSA containing ca 15,OOOcpm of ‘2SI-labelled prolactin (35 pCi/pg protein; Netria) or 1251labelled b-galactosidase [iodinated by the Iodogen method of Salacinski et al. (1981)]; 200 @i/50 pg /I-galactosidase. Samples were incubated at room temperature overnight and the antibody-prolactin or antibody-b-galactosidase complexes were precipitated, washed and counted as described earlier.
RESULTS The cloning procedure used gave rise to several hundred colonies containing recombinant plasmids.
A number of these plasmids were isolated and characterized by restriction enzyme analysis (results not shown) which confirmed that they contained the full-length cDNA for human prolactin. These plasmids were designated pJMBG62. The proteins synthesized by cultures containing plasmid pJMBG62 were analysed by SDS-PAGE. Figure 2(a) shows that cultures of E. coli harbouring pJMBG62 synthesized a protein which migrates with a slower electrophoretic mobility than Bgalactosidase; the molecular weight of this novel protein (140 kDa) compares favourably with the expected size for a /I-galactosidase-prolactin fusion protein. The presence of prolactin epitopes on the fusion protein was tested by Western blot analysis. The results [Fig. 2(b)] indicate that the fusion protein synthesized by E. coli cells harbouring pJMBG62 is detected by the prolactin antibody used (P2). On examination under the phase-contrast microscope, cultures of E. coli harbouring pJMBG62 were found to contain large number of inclusion bodies. When the inclusion bodies were washed as described earlier, some of the fusion protein started to appear in the supematant fraction only when the wash solution contained 5 M urea (results not shown). Therefore, inclusion bodies were washed in 0.1 M Tris-HCl pH 8.0 containing 2.5 M urea. After solubilization in the presence of 8 M urea, >70% of the fusion protein appeared in the soluble fraction. It was observed that during the renaturation process some
M. S. GILBERTer al.
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of the fusion protein formed a precipitate; however, the majority (~70%) remained in the soluble fraction. Using this methodology and starting with a 200 ml culture, a typical purification yielded 9.6 mg of total protein after the final dialysis step. The concentration and immunoreactivity of the prolactin in the renatured fusion protein were determined using a radioimmunoassay capable of detecting prolactin concentrations as low as 50 ng/ml (results not shown). In assays containing the equivalent of 88 pg total protein the radioimmunoassay detected the equivalent of 3.9 pg of prolactin. Therefore, the total amount of prolactin fusion protein present in the above protein preparation (9.2 mg total protein) was 422pg which corresponds to 4.4% of the total recovered protein. This preparation was used as an immunogen to raise antibodies against human prolactin. Analysis of sera from rabbits MGl and MG2 (previously immunized with pure prolactin and reimmunized with the fusion protein) indicated that they showed an immediate response with respect to the presence of antibodies against prolactin (Fig. 3); the highest titres were observed after the second injection and were l/2000 (antibody dilution at which 50% of the total ‘Z51-labelled prolactin was bound; 50% B,) for both rabbits. After subsequent injections, the antibody titres decreased to control levels (results not shown). In the case of rabbits MG3-6 (immunized with the /I-galactosidase-prolactin fusion protein) no prolactin antibodies were produced over a period of three months; the results obtained were comparable with the control sera taken from the rabbits before the first injection. Sera from the first bleeds of rabbits MG7-10 (immunized with pure human prolactin) gave results similar to those obtained with the control sera. 100,
1
60 -
z
s
P
60-
c
F 27
e P
a4
40 -
20 -1
i_ 1st bleed
2nd bleed
3rd bleed
Fig 3. Mean % ‘251-labelled prolactin bound at 1 in 250 dilition in sera from control and from the first three bleeds from rabbits MGl-2 (open bars), MG3-6 (hatched bars) and MG7-10 (solid bars). Vertical lines represent *SEM.
However, following the second and third injections the sera from the four rabbits contained antibodies to human prolactin (Fig. 4). The antibody titres which gave 50% B, ranged between l/2000 and l/4000 (results not shown). The antibody titres for /I-galactosidase were also analysed for all the rabbits (results not shown). Rabbits MGl-6 showed a good response to /I-galactosidase (titres > l/64,000 gave 50% B,) whilst rabbits MG7-10 showed no response. Further confirmation of the presence of /I-galactosidase antibodies was obtained from Western blots using antisera from rabbits MG3-6. These blots not only showed a response to p-galactosidase, but also to smaller E. coli proteins present in the partially-pure /I-galactosidaseprolactin fusion protein. A typical Western blot using antisera from rabbit MG5 is shown in Fig. 4; similar results were obtained with antisera from rabbits MG3, MG4 and MG6.
DISCUSSION The cloning
strategy
and
the expression
vector
(pUR291) used ensured that the prolactin cDNA fragment was inserted in the correct reading frame for expression. Expression of proteins in this vector results in the production of /I-galactosidase fusion proteins in which the foreign protein is fused to the carboxy terminus of /I-galactosidase. The expression of /I-galactosidase fusion proteins in E. coli cells harbouring plasmid pJMBG62 was confirmed by SDS-PAGE [Fig. 2(a)] and by Western blot analysis [Fig. 2(b)]. The fact that the j3-galactosidase-prolactin fusion protein is present in inclusion bodies of E. coli cells harbouring pJMBG62 does not allow the direct use of affinity chromatography procedures for its purification. Such procedures have been described by Carroll and Laughton (1985) and by Ullmann (1984) and have been used previously in this laboratory for the purification of a /I-galactosidase-POMC (59241) fusion protein (Grewal et al., 1989). Therefore. a different purification procedure involving isolation and solubilization of inclusion bodies in 8 M urea was employed. This solubilized > 70% of the fusion protein but the gradual removal of the urea resulted in the precipitation of ca 20% of the solubilized protein. This method was also efficient in removing some contaminating E. coli proteins, thus resulting in a partially-pure preparation of the fusion protein. Using a two antibody radioimmunoassay, the amount of immunoreactive prolactin present as a /3-galactosidase fusion protein in this preparation was found to be 4.4% of the total solubilized protein. The results obtained using the partially-pure flgalactosidase-prolactin fusion protein as an immunogen show that antibodies to prolactin were only produced by the rabbits previously exposed to purified prolactin (rabbits MGl and MG2). When compared with the results obtained using purified prolactin as the immunogen (rabbits MG7-lo), it can be seen that although the partially-pure fl-galactosidase-prolactin fusion protein does not appear to elicit a primary immune response it does produce a sec-
116
A 116
B
Fig. 2. (a) SDS-polyacrylamide gel (stained with Coomassie Blue) showing total cellular protein from E. coli BMH 71-18 (lane 1) and from E. coli BMH 71-18 containing plasmid pUR291 (lane 2) or plasmid pJMBG62 (lane 3). A and B indicate the /?-galactosidase-prolactin fusion protein and /?-galactosidase respectively; molecular weight sizes are shown in kDa. (b) Western blot analysis of total cellular protein from E. coli BMH 71-18 containing plasmid pJMBG62 (lane l), plasmid pUR291 (lane 2) or no plasmid (lane 3). Antibodies used were as stated in Materials and Methods. The position of the fusion protein in lane 1 is indicated by A; molecular weight sizes are shown in kDa.
111
205
116
97
Fig. 4. Western blot analysis using antiserum from rabbit MG5. The samples analysed were pure b-galactosidase (lane 1), total cellular protein from E. coli BMH 71-18 (lane 2), and the partially-pure /S-galactosidase-prolactin fusion protein (lane 3). Molecular weight sizes are shown in kDa; the fusion protein and the /?-galactosidase are indicated by A and B respectively.
112
Human prolactin in E. eofi ondary immune response (Fig. 3).The lack of this primary response may be due to the administration of inadequate amounts of prolactin in the presence of larger amounts of other proteins. In addition, the impurities may detract from the ability of the immune system to produce antibodies specific for the prolactin moiety. Rabbits MG3-6 produced high titre antibodies to /3-galactosidase as demonstrated by the binding of the radioiodinated B-galactosidase; antibodies to other smaller E. coli proteins were also produced as demonstrated by Western blot analysis (Fig. 4). The evidence presented here merits further investigation in order to improve our understanding of the immunogenic properties of prolactin alone or as a fusion protein. The use of fusion proteins containing different portions of human prolactin (such as that encoded by plasmid pJMBG60) will also allow epitope mapping studies to be carried out. In conclusion, this report describes the expression, ch~a~te~zation and partiai purification of a human prolactin fusion protein in E. coli; this fusion protein was used successfully to raise polyclonal antibodies against human prolactin in rabbits previously exposed to human prolactin. Preliminary studies using a pigeon crop sac bioassay and a radioreceptor assay suggest that this partially purified ~-galactosidase human prolactin fusion protein is biologically active (results not shown). This molecular biology approach offers a number of advantages when compared with the conventional method of obtaining human prolactin. Firstly, it provides an unlimited source of a particular hormone. Secondly, it eliminates the need for handling human tissues, thus removing possible exposure to pathogens. Thirdly, the hormone is obtained as a ready-made ‘hapten-carrier’ molecule, hence eliminating the need for coupling reactions which are not always efficient. This approach has been shown to be a valuable tool for raising monoclonal and polyclonal antibodies against prohormones and other related peptides. For example, Spruce et al. (1988) have used it to raise antibodies against proenkephalin A whilst in our laboratory it has been used to raise polyclonal antibodies against the corticotropin releasing hormone (CRI-I) precursor (Castro et al., 1989) and against part of the proopiomelanocortin (POMC) precursor the expression of which has been reported earlier (Grewal et al., 1989). Acknowledgements-We
would like to thank our colleagues
in the Joint Molecular Biology Group and in the Molecular Endocrinology Group for helpful criticisms and advice during this work. Special thanks are due to Mr P. Merrison for helping with the immuni~tion schedule.
REFERENCES
Beck P., Parker M. L. and Daughaday W. H. (1965) Radioimmunologic measurement of human placental lactogen in plasma by a double antibody method during normal and diabetic pregnancies. Clin. Endocr. 25, 1457-14762.
Bimboim H. C. and Doly J. (1979) A rapid alkaline method extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7, 1513-1518. Blackwell R. E. (1985) Diagnosis and management of prolactinomas. Fertil. Steril. 43, 5-16.
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Boyer H. W. (1979) A complementation analysis of the restriction and modification of DNA in E. coli. J. mofec. Biol. 41, 459-464. Carroll S. B. and Laughton A. (1985) Production and purification of polyclonal antibodies to the foreign segment of B-gala&oGdase fusion proteins. In DNA &‘on&: A Practical Approach Vol. III (Edited by Glover D.), pp. 89-l 11. IRL Press, Oxford. Castro M. G., Dils R. C., Saphier P., Linton E. A., Sawa D. and Lowry P. J. (1989) Polvclonal antibodies against recombinant human‘pre-l&&icotropin releasing hormone (h-pre-ProCRH). Proceedings of the Society for Neuroscience. Abstract. 58.10. Catt K. J., Moffat B. and Niall H. D. (1967) Human growth hormone and placental lactogen: structural similarities. Science 157, 321-326. Cooke N. E., Coit D., Shine J., Baxter J. D. and Martial A. (1981) Human prolactin: cDNA structural analysis and evolutionary comparisons. J. biol. Chem. 256, 4007-4016.
Grewal T. S., Lowry P. J. and Sawa, D. (1989) Expression and partial purgation of human pro-opiomelan~ortin in E~cherjehja co&. J. molec. Endocr. 3, 105-112. Grunstein M. and Hogness D. (1975) Colony hybridization. A method for the isolation of cloned DNAs that contain a specific gene. Proc. natn. Acad. Sci. U.S.A. 72, 3961-3967s
Hodgkinson S. C. and Lowry P. J. (1981) Hydrophobic interaction chromatography and anion exchange chromatography in the presence of acetonitrile. Biochem. J. 199, 619-627.
Hodgkinson S. C., Landon J. and Lowry P. J. (1984) A liquid phase two site immunoradiometric assay for prolactin. Biochem. J. 211, 273-279. Mandel M. and Higa A. (1970) Calcium dependent bacteriophage DNA infection. J. molec. Biol. 53, 159162. Marston F. A, 0. (1985) The purification of eukaryotic peptides expressed in Exherichia coli. In DNA Cloning: A Practical Approach Vol. III (Edited by Glover D.), pp. 59-88. IRL Press, Oxford. Messing J., Gronenborn B., Muller-Hill B. and HofSchneider H. P. (1977) Filamentous coliuhaee . - Ml3 as a cloning vehicle: insert&n of a Hind111 fragment of the luc regulatory region in Ml3 replicative form in u&o. Proc. natn. Acad. Sci. U.S.A. 74, 3624-3646.
Moltich M. (1985) Pregnancy and the hyperprolactinaemic woman. N. Engl. J. Med. 312, 1364-1369. Niall H. A., Hog& M. L., Tregar G., Segre G. V., Hwang P. and Friesen H. (1973) The chemistrv of growth hormone and lactogenic ho&one. Rec. Frog. H&m. Res, 29, 387-416. Nickel J. C. (1984) Endocrine dysfunction in impotence. J. Ural. 132, 40-46. Pardee A. B., Jacob F. and Monod J. (1959) The genetic control and cytoplasmic expression of ‘inducibility’ in the synthesis of /3-galacosidase by E. co/i. J. molec. Biol. 1,
165-178. Ruther U. and Muller-Hill B. (1983) Easy identification of cDNA clones. EMBO J 2, 1791-1794. Salacinski P. R. P., McLean C., Sykes J. E. C., ClementJones V. V. and Lowry P. J. (1981) Iodination of proteins, glycoproteins and peptides using a solid-phase oxidizing agent, 1,3,4,6-tetrachloro-3a,6a-diphenyl alycoluril (fodogen). Analyt. Biochem. 117, 136-148. Sawa D. (1983) Cloning and restriction enzvme analysis of a Nei&eria gono&hoeae plasmid. Microbioi 38, 7-14.
Sinha Y. N., Selby F. W., Lewis U. J. and Vanderlaan W. P. (1973) A homologous radioimmunoasssy for human prolactin. J. clin. Endocr. Metab. 36, 509-516. Spruce B., Jackson S., Lowry P. J., Lane D. P. and Glover D. M. (1988) Monoclonal antibodies to proenkephalin A
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fusion peptide synthesized in E. coli recognized novel proenkephalin A precursor forms. J. bid. Chem. 263, 19,788-19,795. Ullmann A. (1984) One step purification of hybrid proteins which have j-galactosidase activity. Gene 29, 27-31. Vogelstein B. and Feinberg A. (1983) A technique for
radiolabelling restriction endonuclease fragments to a high specific activity. Analyt. Biochem. 132, 6-13. Weislander L. (1979) A simple method to recover intact high molecular weight RNA and DNA after electrophoresis separation in low gelling temperature agarose gels. Analyt. Biochem. 98, 305-312.
0020-711X/91
1991
$3.00 + 0.00
Copyright 0 1991Pergamon Press plc
EXPRESSION AND PARTIAL PURIFICATION OF HUMAN PROLACTIN IN ESCHERICHIA COLI M. S. GILBERT, P. J. LOWRY, M. G. CASTRO, R. J. WDDDSand D. SAVVA* School of Animal and Microbial Sciences, Department of Biochemistry & Physiology, University of Reading, Whiteknights, P.O. Box 228, Reading RG6 2AJ, England [Tel. (0734) 875123; Fax (0734) 310180] (Received 3 May 1990)
Abstract-l. Human prolactin has been expressed in Escherichia co/i. A cDNA fragment coding for the signal sequence and the full length prolactin molecule was cloned into the expression vector pUR291 which directs the synthesis of a B-galactosidase prolactin fusion protein when expressed in E. cofi. 2. Cultures of E. coli harbouring the recombinant plasmid pJMBG62 produced a fusion protein of the appropriate molecular weight which was detected by Western blot analysis using a polyclonal antibody raised against pituitary-derived human prolactin. 3. The fusion protein was isolated from inclusion bodies in a partially pure form and it was used as immunogen to raise antibodies against human prolactin. 4. When this partially purified fusion protein was injected into rabbits it generated antisera with good prolactin titres in animals which were rested for one year following a disappointing primary immunization with purified human prolactin.
INTRODUCTION Human prolactin is a single chain polypeptide of 199 amino acids and a molecular weight of 23,000. It is synthesized and released from the lactotrophic cells of the anterior pituitary and belongs to a set of hormones collectively known as the ‘Prl Set’ (Cooke et al., 1981) which includes growth hormone and chorionic somatomammotropin (placental lectogen). It is believed that the ‘Prl Set’ hormones evolved from a common ancestral gene by subsequent sequence divergence (Catt et al., 1967), as indicated by similarities in their amino acid sequences and by overlaps in their biological and immunological properties (Niall et al., 1973). In humans the major area of action of prolactin is on the mammary gland where it is involved in the stimulation of postpartum lactation. It is also involved in other aspects of pregnancy and has some physiological effects in men (Moltich, 1985; Blackwell, 1985). Several disorders have been associated with prolactin. In women, abnormal levels of serum prolactin have been linked with such conditions as galactorrhea, anovulation with amenorrhea, hypooestrogenism and hyperprolactinaemia which has been shown to be common in some 10% of all female infertilities. In men, hyperprolactinaemia has been linked to sexual disorders such as impotence and ejaculatory incompetence (Nickel, 1984). The ability to detect circulating levels of prolactin in patients with the above disorders would be a useful diagnostic tool. The main methods currently employed for the detection of circulating prolactin are radioimmunoassays (Sinha et al., 1973) and immunoradiometric assays (Hodgkinson et al., 1984). Both of these depend on the availability of high specificity *To whom all correspondence Bc2311-P
should be addressed
antiserum to prolactin and these in turn depend on the availability of the human hormone for use as an antigen but its immunogenicity can sometimes be poor. Recent advances in recombinant DNA technology provide a means of producing human prolactin fusion proteins in bacteria such as Escherichiu coli. Also, a molecular biology approach would eliminate the need of handling human tissues and will provide a non-infectious source of prolactin, eliminating contact with viruses such as HIV and hepatitis among others. This paper describes: (1) the expression of human prolactin in E. coli using a pUR expression vector (Ruther and Muller-Hill, 1983); (2) the partial purification of a /I-galactosidase fusion protein containing the full length human prolactin at its carboxy-terminus; and (3) the use of this fusion protein to raise antibodies against human prolactin. MATERIALSAND METHODS Piasmidr and bacterial strains
Human prolactin cDNA cloned into plasmid pBR322 [a generous gift from Dr J. Martial, Institute of Chemistry, Sart Tilman, Belgium; Cooke et al. (1981)] was maintained in E. coli HBlOl (Boyer, 1969). The expression vector pUR291 was supplied by Dr U. Ruther (University of Kiiln, West Germany) and it was maintained in E. co/i BMH 71-18 (Messing et al., 1977). Bacterial strains were transformed using the CaCl, procedure of Mandel and Higa (1970), and transformants were selected on LB (10 g/l Bacto-tryptone. 5 g/l Bacto-yeast extract, 5 g/l NaCl, i g/i’glucose; bacterialogical powders were from Lab M Ltd. Salford. Manchester. U.K.) agar plates containing ampicilhn (50&ml). Large scale plasmid preparations were performed using the method described by Bimboim and Doly (1979) with the modifications described by Sawa (1983). 107
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M. S. GILBERT~~ al.
Construction and identification of recombinant plasmids
Restriction endonucleases and T4 DNA ligase were obtained form Bethesda Research Laboratories (BRL, Paisley, Strathclyde, U.K.) and were used as recommended by the supplier. b-galactosidase was supplied by Sigma Chemical Company Ltd. (Poole, Dorset, U.K.). DNA restriction fragments were separated on 0.8% (w/v) agarose gels in TBE (89 mM Tris base, 90 mM boric acid, 1 mM Na, EDTA, pH 8.0). When necessary, fragments were purified from low melting point agarose gels (Weislander, 1979). The recombinant pBR322 plasmid containing the human prolactin cDNA [Fig. l(a)] was cleaved with PstI to generate three DNA fragments: the pBR322 vector and two smaller fragments of 588 and 367 bp. The 588 bp fragment codes for 10 glycines, an asparagine, the 28 amino acids of the signal sequence and the first 157 amino acids of human prolactin; this was ligated with PsrI-cleaved expression vector pUR291 to generate plasmid pJMBG60 [Fig. l(b)]. This plasmid was used to construct an expression plasmid containing the complete cDNA for human prolactin. Briefly, after digestion with BglII and Hind111 the 70 bp fragment was removed and replaced with the 222 bp BglII-Hind111 fragment from the original prolactin cDNA [Fig. l(a)]. The ligation mixture was used to transform E. co/i BMH 71-18. Plasmid DNA was isolated from some of the transformants and analysed with restriction enzymes. Recombinant plasmids were identified by in situ colony hybridization (Grunstein and Hogness, 1975). Bacterial colonies were transferred from the agar plates onto
Hybond-N membranes as described by the supplier of the membrane (Amersham International plc, Amersham, Bucks., U.K.). Transformants were screened using the 700 bp AoaII-AuaII fragment of the prolactin cDNA [Fig. l(a)] which was labelled to a sp. act. of 2 x lo9 dpm/pg DNA using [a-32P]dCTP (3000 Ci/mmol; Amersham International) and the method of Vogelstein and Feinberg (1983). Hybridization was performed at 65°C overnight in 6 x SSC (0.9 M NaCl, 0.09 M trisodium citrate, pH 7.0) containing 0.5% (w/v) dried skimmed milk. Membranes were washed at 65°C using 0.1 x SSC (15 mM NaCl, 1.5 mM trisodium citrate, pH 7.0) containing 0.1% (w/v) sodium dodecyl sulphate (SDS) and were autoradiographed. Analysis of recombinant proteins by SDS-polyacrylamiak electrophoresis and Western blotting
Cultures harbouring recombinant plasmids were grown and processed for SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting as described by Grewal et al. (1989). Protein concentrations were determined by a Coomassie Blue assay using Pierce Reagent (Pierce Chemical Co., Rockford, Ill., U.S.A.). The activity of b-galactosidase was determined as described previously (Pardee et al.. 1959). The primary antibody used for the Western blots was a rabbit anti-human prolactin antibody (P2) and the second antibody was a peroxidase-conjugated goat anti-rabbit IgG Fc fragment specific antibody (Jackson Immunoresearch Laboratories Inc., Pa, U.S.A.). Antibody complexes were detected by the appearance of coloured bands after the membranes were incubated in 100 ml PBS (10 mM sodium
(a)
-28
PSJ
gel
+I
4!Ld
100 bp
(b)
Fig. 1. (a) Partial restriction map of the human prolactin cDNA. The solid, open and hatched boxes represent the signal sequence, the coding sequence and the 3’ untranslated cDNA respectively. The solid lines indicate homopolymer tails. Numbers refer to amino acid residues. (b) Schematic diagram and partial restriction map of plasmids pJMBG60 and pJMBG62 containing different portions of human prolactin cDNA (not drawn to scale). For the cDNA closed, open and hatched boxes as in Fig. l(a). The /I-galactosidase (la&) and the ampicillin resistance (ApR) genes on the plasmid are also shown.
Human prolactin in E. coli phospahte buffer pH 7.5, 0.13 M sodium chloride) containing 20mg of 3,3-diaminobenzidine (Sigma Chemical Co., Poole, Dorset, U.K.) and 10~1 H,O, (100~01). All incubations were carried out at room temperature with gentle shaking. Isolation of inclusion bodies
Inclusion bodies produced by cultures containing plasmid pJMBG62 were isolated according to Marston (1985); in order to ensure complete lysis of the cells, an additional sonication step was introduced (three 30 set bursts). The cell lysate was centrifuged at 10,OOOgfor 15 min, the supernatant was removed and the pellet containing the inclusion bodies was resuspended in 0.04~01 lysis buffer (50mM Tris-HCl pH 8.0, 1 mM Na, EDTA pH 8.0, 0.1 M NaCl) containing 0.5% (v/v) Triton X-100. After incubation for 30 min at room temperature, the sample was centrifuged as before and the pellets washed in 0.1 ~010.1 M Tris-HCl pH 8.0 containing different amounts of urea (0.1 M-5.0 M) in order to determine the highest concentration of urea at which the fusion protein was still insoluble but which would remove as many E. coli contaminating proteins as possible. Aliquots from each stage were analysed by electrophoresis on SDSpolyacrylamide gels. Purification, solubilization and renaturation of fusion protein
Cell pellets containing inclusion bodies were first washed with lysis buffer containing 0.5% (v/v) Triton X-100 and then with 0.1 M Tris-HCl-pH 8.0.containing 2.5 M urea before solubilization in 0.1 vol 0.1 M Tris-HCl DH 8.0 containing 8 M urea; samples were incubated at room temperature for 20 min and centrifuged at 10,OOOg for 30 min. The urea was removed from the samples by means of serial dilutions in 50 mM Tris-HCl pH 8.0, 1mM Na, EDTA pH 8.0, 100mM NaCl, 0.5% (v/v) 2-mercaptoethanol, 20% (v/v) glycerol as follows: an equal volume of dilution buffer was added dropwise to the samples and mixed gently for 3 hr at 4°C to reduce the urea concentration to 2 M before dialysis of the samples for 24 hr at 4°C against 50 mM Tris-HCl pH 8.0, 1 mM Na, EDTA, 0.5% (v/v) 2-mercaptoethanol, 20% (v/v) glycerol. Radioimmunoassay for human prolactin The two-antibody method described by Beck et al. (1965) was used. The total volume of 250 ~1 consisted of 100 ~1 of a l/500 dilution of the prolactin antibody (P2), 50~1 1251-labelled nrolactin (ca 15.000 cnm: Netria. 36 uCi/u e protein) and 100 ~1 of prolactin Astandards ‘(Netria,’ ‘ST Bartholomew’s Hospital, London), ranging in concentration from 800 to 0.78 ng/ml, or dilutions of the samples to be tested. All dilutions were made up in 50 mM sodium phosphate buffer pH 7.4 containing 0.5% (w/v) bovine serum albumin. Samples were incubated for 24 hr at room temperature before precipitation (30 min at room temperature) with 200 ~1 of precipitating solution (50 mM sodium phosphate buffer pH 7.4, 10% v/v sheep anti-rabbit Fc antiserum, 4% w/v polyethylene glycol6000, 1% v/v normal rabbit serum; this solution was incubated at 4°C for 24 hr prior to use). The sheep anti-rabbit Fc antiserum was obtained from Polvclonal Antibodies Ltd (Dvfed. Wales). After addition of -2 ml 0.15 M NaCl, samples were centrifuged at 2500 g for 20 min at 4”C, the supematants were removed and the radioactivity in the pellets was determined on a LKB gamma counter. Immunization of rabbits
A total of 10 rabbits (MGl-MGlO, New Zealand white, 2 kg in weight; Regal Rabbits, Great Bookham, Surrey, U.K.) were employed for this experiment. Two of the rabbits (MGI and MG2) had previously been iniected over a period of a year with purified natural human prolactin (Hodgkinson et al., 1984) and had been rested for 1 year.
109
All rabbits were primed by intramuscular injections at three sites with a mixture consisting of 3 ml Freund’s complete adjuvant (except in the case of rabbits MGI and MG2 where the mixture contained Freund’s incomplete adjuvant), 5~1 Brij’s solution (30% v/v; Sigma) and 1 ml saline containing the required amount of the immunogen. The immunogen used was 2.3 mg partiallypure prolactin fusion protein (equivalent to 1OOpg pure nrolactin) for rabbits MGl-6 and 50 tirr of purified human prolactin’ for rabbits MG7-10. Subsequent injections (at monthly intervals) were similar except that Freund’s incomplete adjuvant was used and the amount of immunogen was halved. Bleeds (20 ml) were taken from the ear of each rabbit 10 days after each injection and the clots were allowed to form an contract at room temperature for 24 hr. The sera were removed and subsequently stored at -20°C. Prolactin and /I-galactosidase antibody titres were assayed by measuring the amount of 12SI-labelled prolactin and 1251-labelled/Igalactosidase bound by a series of semm dilutions ranging from l/250 to l/64,000. Each assay had a total volume of 200~1, consisting of 100~1 antibody (diluted in 50mM sodium phosphate buffer pH 7.4, 0.5% w/v bovine serum albumin) and 100 ~1 of 50mM sodium phosphate buffer pH 7.4, 0.5% (w/v) BSA containing ca 15,OOOcpm of ‘2SI-labelled prolactin (35 pCi/pg protein; Netria) or 1251labelled b-galactosidase [iodinated by the Iodogen method of Salacinski et al. (1981)]; 200 @i/50 pg /I-galactosidase. Samples were incubated at room temperature overnight and the antibody-prolactin or antibody-b-galactosidase complexes were precipitated, washed and counted as described earlier.
RESULTS The cloning procedure used gave rise to several hundred colonies containing recombinant plasmids.
A number of these plasmids were isolated and characterized by restriction enzyme analysis (results not shown) which confirmed that they contained the full-length cDNA for human prolactin. These plasmids were designated pJMBG62. The proteins synthesized by cultures containing plasmid pJMBG62 were analysed by SDS-PAGE. Figure 2(a) shows that cultures of E. coli harbouring pJMBG62 synthesized a protein which migrates with a slower electrophoretic mobility than Bgalactosidase; the molecular weight of this novel protein (140 kDa) compares favourably with the expected size for a /I-galactosidase-prolactin fusion protein. The presence of prolactin epitopes on the fusion protein was tested by Western blot analysis. The results [Fig. 2(b)] indicate that the fusion protein synthesized by E. coli cells harbouring pJMBG62 is detected by the prolactin antibody used (P2). On examination under the phase-contrast microscope, cultures of E. coli harbouring pJMBG62 were found to contain large number of inclusion bodies. When the inclusion bodies were washed as described earlier, some of the fusion protein started to appear in the supematant fraction only when the wash solution contained 5 M urea (results not shown). Therefore, inclusion bodies were washed in 0.1 M Tris-HCl pH 8.0 containing 2.5 M urea. After solubilization in the presence of 8 M urea, >70% of the fusion protein appeared in the soluble fraction. It was observed that during the renaturation process some
M. S. GILBERTer al.
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of the fusion protein formed a precipitate; however, the majority (~70%) remained in the soluble fraction. Using this methodology and starting with a 200 ml culture, a typical purification yielded 9.6 mg of total protein after the final dialysis step. The concentration and immunoreactivity of the prolactin in the renatured fusion protein were determined using a radioimmunoassay capable of detecting prolactin concentrations as low as 50 ng/ml (results not shown). In assays containing the equivalent of 88 pg total protein the radioimmunoassay detected the equivalent of 3.9 pg of prolactin. Therefore, the total amount of prolactin fusion protein present in the above protein preparation (9.2 mg total protein) was 422pg which corresponds to 4.4% of the total recovered protein. This preparation was used as an immunogen to raise antibodies against human prolactin. Analysis of sera from rabbits MGl and MG2 (previously immunized with pure prolactin and reimmunized with the fusion protein) indicated that they showed an immediate response with respect to the presence of antibodies against prolactin (Fig. 3); the highest titres were observed after the second injection and were l/2000 (antibody dilution at which 50% of the total ‘Z51-labelled prolactin was bound; 50% B,) for both rabbits. After subsequent injections, the antibody titres decreased to control levels (results not shown). In the case of rabbits MG3-6 (immunized with the /I-galactosidase-prolactin fusion protein) no prolactin antibodies were produced over a period of three months; the results obtained were comparable with the control sera taken from the rabbits before the first injection. Sera from the first bleeds of rabbits MG7-10 (immunized with pure human prolactin) gave results similar to those obtained with the control sera. 100,
1
60 -
z
s
P
60-
c
F 27
e P
a4
40 -
20 -1
i_ 1st bleed
2nd bleed
3rd bleed
Fig 3. Mean % ‘251-labelled prolactin bound at 1 in 250 dilition in sera from control and from the first three bleeds from rabbits MGl-2 (open bars), MG3-6 (hatched bars) and MG7-10 (solid bars). Vertical lines represent *SEM.
However, following the second and third injections the sera from the four rabbits contained antibodies to human prolactin (Fig. 4). The antibody titres which gave 50% B, ranged between l/2000 and l/4000 (results not shown). The antibody titres for /I-galactosidase were also analysed for all the rabbits (results not shown). Rabbits MGl-6 showed a good response to /I-galactosidase (titres > l/64,000 gave 50% B,) whilst rabbits MG7-10 showed no response. Further confirmation of the presence of /I-galactosidase antibodies was obtained from Western blots using antisera from rabbits MG3-6. These blots not only showed a response to p-galactosidase, but also to smaller E. coli proteins present in the partially-pure /I-galactosidaseprolactin fusion protein. A typical Western blot using antisera from rabbit MG5 is shown in Fig. 4; similar results were obtained with antisera from rabbits MG3, MG4 and MG6.
DISCUSSION The cloning
strategy
and
the expression
vector
(pUR291) used ensured that the prolactin cDNA fragment was inserted in the correct reading frame for expression. Expression of proteins in this vector results in the production of /I-galactosidase fusion proteins in which the foreign protein is fused to the carboxy terminus of /I-galactosidase. The expression of /I-galactosidase fusion proteins in E. coli cells harbouring plasmid pJMBG62 was confirmed by SDS-PAGE [Fig. 2(a)] and by Western blot analysis [Fig. 2(b)]. The fact that the j3-galactosidase-prolactin fusion protein is present in inclusion bodies of E. coli cells harbouring pJMBG62 does not allow the direct use of affinity chromatography procedures for its purification. Such procedures have been described by Carroll and Laughton (1985) and by Ullmann (1984) and have been used previously in this laboratory for the purification of a /I-galactosidase-POMC (59241) fusion protein (Grewal et al., 1989). Therefore. a different purification procedure involving isolation and solubilization of inclusion bodies in 8 M urea was employed. This solubilized > 70% of the fusion protein but the gradual removal of the urea resulted in the precipitation of ca 20% of the solubilized protein. This method was also efficient in removing some contaminating E. coli proteins, thus resulting in a partially-pure preparation of the fusion protein. Using a two antibody radioimmunoassay, the amount of immunoreactive prolactin present as a /3-galactosidase fusion protein in this preparation was found to be 4.4% of the total solubilized protein. The results obtained using the partially-pure flgalactosidase-prolactin fusion protein as an immunogen show that antibodies to prolactin were only produced by the rabbits previously exposed to purified prolactin (rabbits MGl and MG2). When compared with the results obtained using purified prolactin as the immunogen (rabbits MG7-lo), it can be seen that although the partially-pure fl-galactosidase-prolactin fusion protein does not appear to elicit a primary immune response it does produce a sec-
116
A 116
B
Fig. 2. (a) SDS-polyacrylamide gel (stained with Coomassie Blue) showing total cellular protein from E. coli BMH 71-18 (lane 1) and from E. coli BMH 71-18 containing plasmid pUR291 (lane 2) or plasmid pJMBG62 (lane 3). A and B indicate the /?-galactosidase-prolactin fusion protein and /?-galactosidase respectively; molecular weight sizes are shown in kDa. (b) Western blot analysis of total cellular protein from E. coli BMH 71-18 containing plasmid pJMBG62 (lane l), plasmid pUR291 (lane 2) or no plasmid (lane 3). Antibodies used were as stated in Materials and Methods. The position of the fusion protein in lane 1 is indicated by A; molecular weight sizes are shown in kDa.
111
205
116
97
Fig. 4. Western blot analysis using antiserum from rabbit MG5. The samples analysed were pure b-galactosidase (lane 1), total cellular protein from E. coli BMH 71-18 (lane 2), and the partially-pure /S-galactosidase-prolactin fusion protein (lane 3). Molecular weight sizes are shown in kDa; the fusion protein and the /?-galactosidase are indicated by A and B respectively.
112
Human prolactin in E. eofi ondary immune response (Fig. 3).The lack of this primary response may be due to the administration of inadequate amounts of prolactin in the presence of larger amounts of other proteins. In addition, the impurities may detract from the ability of the immune system to produce antibodies specific for the prolactin moiety. Rabbits MG3-6 produced high titre antibodies to /3-galactosidase as demonstrated by the binding of the radioiodinated B-galactosidase; antibodies to other smaller E. coli proteins were also produced as demonstrated by Western blot analysis (Fig. 4). The evidence presented here merits further investigation in order to improve our understanding of the immunogenic properties of prolactin alone or as a fusion protein. The use of fusion proteins containing different portions of human prolactin (such as that encoded by plasmid pJMBG60) will also allow epitope mapping studies to be carried out. In conclusion, this report describes the expression, ch~a~te~zation and partiai purification of a human prolactin fusion protein in E. coli; this fusion protein was used successfully to raise polyclonal antibodies against human prolactin in rabbits previously exposed to human prolactin. Preliminary studies using a pigeon crop sac bioassay and a radioreceptor assay suggest that this partially purified ~-galactosidase human prolactin fusion protein is biologically active (results not shown). This molecular biology approach offers a number of advantages when compared with the conventional method of obtaining human prolactin. Firstly, it provides an unlimited source of a particular hormone. Secondly, it eliminates the need for handling human tissues, thus removing possible exposure to pathogens. Thirdly, the hormone is obtained as a ready-made ‘hapten-carrier’ molecule, hence eliminating the need for coupling reactions which are not always efficient. This approach has been shown to be a valuable tool for raising monoclonal and polyclonal antibodies against prohormones and other related peptides. For example, Spruce et al. (1988) have used it to raise antibodies against proenkephalin A whilst in our laboratory it has been used to raise polyclonal antibodies against the corticotropin releasing hormone (CRI-I) precursor (Castro et al., 1989) and against part of the proopiomelanocortin (POMC) precursor the expression of which has been reported earlier (Grewal et al., 1989). Acknowledgements-We
would like to thank our colleagues
in the Joint Molecular Biology Group and in the Molecular Endocrinology Group for helpful criticisms and advice during this work. Special thanks are due to Mr P. Merrison for helping with the immuni~tion schedule.
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