Biologically Active Recombinant Human Progastrin6–80Contains a Tightly Bound Calcium Ion (2001)

Evidence is accumulating that gastrin precursors may act as growth factors for the colonic mucosa in vivo. The aims of this study were to prepare recombinant human progastrin6–80 and to investigate its structure and biological activities in vitro. Human progastrin6–80 was expressed in Escherichia coli as a glutathione S-transferase fusion protein. After thrombin cleavage progastrin6–80 was purified by reverse phase high pressure liquid chromatography and characterized by radioimmunoassay, amino acid sequencing, and mass spectrometry. Assays for metal ions by atomic emission spectroscopy revealed the presence of a single tightly bound calcium ion. Progastrin6–80 at concentrations in the pm to nm range stimulated proliferation of the conditionally transformed mouse colon cell line YAMC. The observations that progastrin6–80 did not bind to either the cholecystokinin (CCK)-A or the gastrin/CCK-B receptor expressed in COS cells and that antagonists selective for either receptor did not reverse the proliferative effects of progastrin6–80suggested that progastrin6–80 stimulated proliferation independently of either the CCK-A or the gastrin/CCK-B receptor. We conclude that recombinant human progastrin6–80 is biologically active and contains a single calcium ion. With the exception of the well known zinc-dependent polymerization of insulin and proinsulin, this is the first report of selective, high affinity binding of metal ions to a prohormone. Evidence is accumulating that gastrin precursors may act as growth factors for the colonic mucosa in vivo. The aims of this study were to prepare recombinant human progastrin6–80 and to investigate its structure and biological activities in vitro. Human progastrin6–80 was expressed in Escherichia coli as a glutathione S-transferase fusion protein. After thrombin cleavage progastrin6–80 was purified by reverse phase high pressure liquid chromatography and characterized by radioimmunoassay, amino acid sequencing, and mass spectrometry. Assays for metal ions by atomic emission spectroscopy revealed the presence of a single tightly bound calcium ion. Progastrin6–80 at concentrations in the pm to nm range stimulated proliferation of the conditionally transformed mouse colon cell line YAMC. The observations that progastrin6–80 did not bind to either the cholecystokinin (CCK)-A or the gastrin/CCK-B receptor expressed in COS cells and that antagonists selective for either receptor did not reverse the proliferative effects of progastrin6–80suggested that progastrin6–80 stimulated proliferation independently of either the CCK-A or the gastrin/CCK-B receptor. We conclude that recombinant human progastrin6–80 is biologically active and contains a single calcium ion. With the exception of the well known zinc-dependent polymerization of insulin and proinsulin, this is the first report of selective, high affinity binding of metal ions to a prohormone. cholecystokinin glutathione S-transferase high pressure liquid chromatography phosphate-buffered saline Gastrin is a classical gut peptide hormone that was identified originally as a stimulant of gastric acid secretion. Like many other peptide hormones, gastrin is synthesized as a large precursor molecule of 101 amino acids (Fig. 1), which is converted to progastrin (80 amino acids) by cleavage of the N-terminal signal peptide. Progastrin is processed further by endo- and carboxypeptidases and by C-terminal amidation to yield the final end products glycine-extended gastrin17 and amidated gastrin17 (1Dockray G.J. Varro A. Dimaline R. Physiol. Rev. 1996; 76: 767-798Crossref PubMed Scopus (86) Google Scholar). Although amidated gastrins were thought originally to be the only forms of the hormone with biological activity, glycine-extended gastrin17 has been shown to stimulate the proliferation of several cell lines (2Seva C. Dickinson C.J. Yamada T. Science. 1994; 265: 410-412Crossref PubMed Scopus (315) Google Scholar, 3Singh P. Owlia A. Varro A. Dai B. Rajaraman S. Wood T. Cancer Res. 1996; 56: 4111-4115PubMed Google Scholar, 4Hollande F. Imdahl A. Mantamadiotis T. Ciccotosto G.D. Shulkes A. Baldwin G.S. Gastroenterology. 1997; 113: 1576-1588Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Progastrin itself appears to act as a growth factor for normal colon, because transgenic mice expressing progastrin in the liver have increased concentrations of serum progastrin and a hyperplastic colonic mucosa (5Wang T.C. Koh T.J. Varro A. Cahill R.J. Dangler C.A. Fox J.G. Dockray G.J. J. Clin. Invest. 1996; 98: 1918-1929Crossref PubMed Scopus (260) Google Scholar). In addition, the observation of increased numbers of aberrant crypt foci (6Singh P. Velasco M. Given R. Wargovich M. Varro A. Wang T.C. Am. J. Physiol. 2000; 278: G390-G399Crossref PubMed Google Scholar) and tumors (7Singh P. Velasco M. Given R. Varro A. Wang T.C. Gastroenterology. 2000; 119: 162-171Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) in the colonic mucosa of transgenic mice overexpressing progastrin in comparison with wild-type mice following treatment with azoxymethane suggests that progastrin may act as a co-carcinogen in the development of colorectal carcinoma. However, the possibility should be borne in mind that progastrin, or a breakdown product, might have been acting indirectly on a tissue other than the colonic mucosa to release a second growth factor responsible for the effects observed in the colon. The possibility that colorectal carcinoma cells might utilize progastrin or progastrin-derived peptides as autocrine growth factors has recently received considerable attention (8Baldwin G.S. Shulkes A. Gut. 1998; 42: 581-584Crossref PubMed Scopus (76) Google Scholar). The autocrine model predicts that a cell synthesizes a particular growth factor, which, after release into the surrounding medium, binds to specific receptors on the surface of the same cell and stimulates the proliferation of that cell. The observation that expression of antisense gastrin mRNA inhibits proliferation of colon-derived cell lines in vitro and in vivo (3Singh P. Owlia A. Varro A. Dai B. Rajaraman S. Wood T. Cancer Res. 1996; 56: 4111-4115PubMed Google Scholar, 4Hollande F. Imdahl A. Mantamadiotis T. Ciccotosto G.D. Shulkes A. Baldwin G.S. Gastroenterology. 1997; 113: 1576-1588Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) provides strong evidence that progastrin or progastrin-derived peptides may act as autocrine growth factors in colorectal carcinoma. As predicted by the autocrine model, most colon carcinomas and derived cell lines synthesize gastrin mRNA and progastrin-derived peptides (see Ref. 8Baldwin G.S. Shulkes A. Gut. 1998; 42: 581-584Crossref PubMed Scopus (76) Google Scholar for review) and increased concentrations of progastrin-derived peptides have been detected in the sera of patients with colorectal carcinoma (9Ciccotosto G.D. McLeish A. Hardy K.J. Shulkes A. Gastroenterology. 1995; 109: 1142-1153Abstract Full Text PDF PubMed Scopus (183) Google Scholar). However, the identity of gastrin receptors on colorectal carcinomas is still unclear (see Ref. 8Baldwin G.S. Shulkes A. Gut. 1998; 42: 581-584Crossref PubMed Scopus (76) Google Scholar for review). Experiments on the role of progastrin and its receptors in the development of colorectal carcinoma have been limited by the scarcity of the prohormone. Small amounts of progastrin1–80 (less than 1 nmol/gm tissue) have been isolated from human tissues (10Rehfeld J.F. Johnsen A.H. Eur. J. Biochem. 1994; 223: 765-773Crossref PubMed Scopus (50) Google Scholar), but the largest progastrin-derived peptide available in bulk to date via organic synthesis is progastrin20–71 (10Rehfeld J.F. Johnsen A.H. Eur. J. Biochem. 1994; 223: 765-773Crossref PubMed Scopus (50) Google Scholar). The related prohormone procholecystokinin (pro-CCK)1 has been expressed with an N-terminal histidine tag and purified from baculovirus-infected insect cells (11Wang W. Yum L. Beinfeld M.C. Peptides. 1997; 18: 1295-1299Crossref PubMed Scopus (12) Google Scholar). We have now developed a method for expression and purification of progastrin6–80 from Escherichia coli, to test directly its biological activities on colon-derived cell lines in vitro, to measure its affinity for gastrin and CCK receptors, and to investigate its structure. Previous structural studies on progastrin-derived peptides have been limited to investigation of the conformation of gastrin17and shorter fragments by circular dichroism and ultraviolet and NMR spectroscopy (Refs. 12Peggion E. Mammi S. Palumbo M. Moroder L Wunsch E. Biopolymers. 1983; 22: 2443-2457Crossref PubMed Scopus (25) Google Scholar, 13Peggion E. Mammi S. Palumbo M. Moroder L Wunsch E. Biopolymers. 1984; 23: 1225-1240Crossref PubMed Scopus (15) Google Scholar, 14Torda A.E. Baldwin G.S. Norton R.S. Biochemistry. 1985; 24: 1720-1727Crossref PubMed Scopus (26) Google Scholar and references therein). Binding of three Mg2+ or Ca2+ ions to human [Nle11]-gastrin13 and [Nle15]-gastrin17 was observed in trifluoroethanol (12Peggion E. Mammi S. Palumbo M. Moroder L Wunsch E. Biopolymers. 1983; 22: 2443-2457Crossref PubMed Scopus (25) Google Scholar, 13Peggion E. Mammi S. Palumbo M. Moroder L Wunsch E. Biopolymers. 1984; 23: 1225-1240Crossref PubMed Scopus (15) Google Scholar), but the affinities are presumably considerably lower in aqueous solution because no binding was detected by either circular dichroism (12Peggion E. Mammi S. Palumbo M. Moroder L Wunsch E. Biopolymers. 1983; 22: 2443-2457Crossref PubMed Scopus (25) Google Scholar) or NMR spectroscopy (14Torda A.E. Baldwin G.S. Norton R.S. Biochemistry. 1985; 24: 1720-1727Crossref PubMed Scopus (26) Google Scholar). In trifluoroethanol the dissociation constants for binding of Ca2+ to the three sites in [Nle15]-gastrin17 wereK 1 = 0.29 μm,K 2 = 0.29 μm, andK 3 = 7.1 μm. To determine whether the N- and C-terminal extensions of progastrin6–80increased its affinity for metal ions, samples of recombinant human progastrin6–80 were analyzed by inductively coupled plasma atomic emission spectroscopy. Here we report that progastrin6–80 is biologically active and contains a single tightly bound calcium ion. With the exception of the well known zinc-dependent polymerization of insulin and proinsulin (15Blundell T.L. Dodson G.G. Dodson E. Hodgkin D.C. Vijayan M. Recent Prog. Horm. Res. 1971; 27: 1-40PubMed Google Scholar), this is the first report of selective, high affinity binding of a metal ion to a prohormone. Gastrin17gly was custom synthesized by Auspep (Melbourne, Australia). The conditionally transformed mouse colon cell line YAMC (16Whitehead R.H. VanEeden P.E. Noble M.D. Ataliotis P. Jat P.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 587-591Crossref PubMed Scopus (339) Google Scholar) was generously provided by Dr. R. H. Whitehead (Ludwig Institute for Cancer Research, Melbourne, Australia). AHindIII-HindIII fragment of human gastrin cDNA, corresponding to nucleotides 59–325 of the sequence reported by Boel and coworkers (17Boel E. Vuust J. Norris F. Norris K. Wind A. Rehfeld J.F. Marcker K.A. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2866-2869Crossref PubMed Scopus (154) Google Scholar) and hence encoding the entire sequence of mature human progastrin1–80 (10Rehfeld J.F. Johnsen A.H. Eur. J. Biochem. 1994; 223: 765-773Crossref PubMed Scopus (50) Google Scholar), was subcloned intoHindIII-cut and dephosphorylated pGEX-2TH (18Smith D.B. Johnson K.S. Gene ( Amst. ). 1988; 67: 31-40Crossref PubMed Scopus (5047) Google Scholar). Clones with the insert in the correct orientation were selected by restriction mapping. The predicted sequence of the fusion protein, which was confirmed by nucleotide sequencing, consisted of glutathioneS-transferase (GST) joined to progastrin1–80 by a 6-amino acid linker (GSEFQA) arising from the multiple cloning site. The GST-progastrin fusion protein was purified from sarkosyl lysates ofE. coli by binding to glutathione-agarose as described by Frangioni and Neel (19Frangioni J.V. Neel B.G. Anal. Biochem. 1993; 210: 179-187Crossref PubMed Scopus (833) Google Scholar). Briefly, E. coli strain NM522 was transformed with the plasmid of interest and grown overnight at 37 °C with shaking in LB medium containing 100 μg/ml of ampicillin. The overnight culture (40 ml) was used to inoculate the same medium (360 ml). When an absorbance at 600 nm of 0.8 was reached, the expression of the GST-progastrin fusion protein was induced by treatment with 0.1 mm isopropylthiogalactoside for 6 h. The bacterial cells were harvested by centrifugation at 2500 ×g for 10 min. The cell pellet was washed in cold STE buffer (10 mm Tris/HCl, pH 8.0, 150 mm NaCl, 1 mm EDTA), and resuspended in 24 ml of STE buffer containing 100 μg/ml lysozyme. After incubation on ice for 15 min dithiothreitol was added to 5 mm, and proteins were solubilized with 1.5% Sarkosyl (Sigma). After vortexing for 15 s, cells were sonicated for 2 × 30 s (power level, 4; duty cycle, 50%) in a model 250 sonifier (Branson Sonic Power Co., Danbury, CT). The lysate was clarified by centrifugation at 2500 × g for 5 min at 4 °C. The supernatant was transferred to a new tube, and Triton X-100 was added to 2%. After vortexing for 10 s, washed glutathione-agarose beads (2 ml/10 ml lysate, 50% (v/v) suspension in phosphate-buffered saline (PBS)) were added, and the suspension was gently mixed by rotation at 4 °C for 1 h. The beads were then washed three times with ice-cold PBS by repeated low speed centrifugation and resuspension in PBS. Finally the beads with the GST-progastrin fusion protein attached were stored at −70 °C in storage buffer (1 ml/1 ml of beads, 50 mm Na+Hepes, pH 7.4, 150 mm NaCl, 5 mmdithiothreitol, 10% (v/v) glycerol). The protein content of samples at various stages of the purification was analyzed by SDS-polyacrylamide gel electrophoresis on 10% gels with a buffer system designed for peptides and proteins in the molecular mass range 5–70 kDa (20Gallagher S.R. Coligan J.E. Dunn B.M. Ploegh H.L. Speicher D.W. Wingfield P.T. Current Protocols in Protein Science. John Wiley & Sons, New York1988: 10.1.11-10.1.12Google Scholar). Progastrin was cleaved from the GST-progastrin fusion protein bound to glutathione-agarose beads by incubation with thrombin (8 units, Sigma) in cleavage buffer (1 ml/1 ml of beads, 50 mm Hepes, pH 8.0, 150 mm NaCl, 2.5 mm CaCl2) for 1 h at 37 °C. Following cleavage, the supernatant containing progastrin was separated from the beads by centrifugation. The beads were washed with elution buffer (1 ml/1 ml of beads, 1 m urea, 50 mm Hepes, pH 7.5), and the washes were combined with the initial supernatant. The recombinant human progastrin prepared above was applied to a C18 column (8 × 100 mm, Waters Associates, Milford, MA), which had been equilibrated with 50 mm ammonium bicarbonate, 20% acetonitrile. The progastrin was eluted with a gradient from 20–50% acetonitrile in 50 mm ammonium bicarbonate at a flow rate of 1 ml/min. Fractions of 0.5 ml were collected and dried on a Speed Vac (Savant, Hicksville, NY) for radioimmunoassay, mass spectrometry, and amino acid sequencing. The concentrations of recombinant human progastrin in chromatographic fractions were measured by radioimmunoassay as previously described (9Ciccotosto G.D. McLeish A. Hardy K.J. Shulkes A. Gastroenterology. 1995; 109: 1142-1153Abstract Full Text PDF PubMed Scopus (183) Google Scholar). A polyclonal antiserum (1137) was raised in rabbits against an undecapeptide consisting of the C-terminal gastrin decapeptide with an additional tyrosine residue at the N terminus for iodination (9Ciccotosto G.D. McLeish A. Hardy K.J. Shulkes A. Gastroenterology. 1995; 109: 1142-1153Abstract Full Text PDF PubMed Scopus (183) Google Scholar). A C-terminal flanking peptide standard curve was constructed with 125I-C-terminal flanking peptide as label. The ID50 was 1.3 ± 0.2 fmol/tube, and the intra-assay variation was <7%. Electrospray ionization mass spectrometry was performed on a Sciex API-300 triple quadrupole mass spectrometer (PerkinElmer Life Sciences) fitted with a micro-ionspray ion source (flow rate, 0.2 μl/min), previously calibrated to an accuracy of ±0.01% using singly charged poly(propylene glycol) reference ions. Samples (2–4 μl) from reverse phase HPLC fractions were mixed with 1:1 acetonitrile, 0.2% formic acid prior to analysis. Signal-averaged mass spectra obtained from 50–100 scans over 5–10 min using anm/z scan range of 100–3000 daltons/unit charge (Da/z) (half-height peak width, 0.6 Da/z) were subsequently analyzed using Sciex BioMultiview software (PerkinElmer Life Sciences). N-terminal amino acid sequences were obtained by sequential Edman degradation using a Hewlett-Packard G1005A automated protein sequencing system, calibrated with phenylthiohydantoin-derivative standards prior to each sequencing run. Samples of recombinant human progastrin from reverse phase HPLC were concentrated on a Speed Vac to remove acetonitrile, treated with 10 mm EDTA in 8 m urea for 16 h at 4 °C, transferred to SpectraPor dialysis tubing (molecular mass cut-off, 3.5 kDa; Spectrum Medical Industries, Houston, TX), and dialyzed against 10 mm Na-Hepes, pH 7.6, containing 10 μm EDTA and 0.005% Tween 20 for 6 days at 4 °C. Control samples were treated in parallel except that the EDTA/urea treatment was omitted. Samples of dialyzed progastrin and of the dialysis buffers were analyzed in triplicate for the presence of aluminum, calcium, cobalt, chromium, copper, iron, magnesium, manganese, nickel, scandium, titanium, vanadium, and zinc by inductively coupled plasma atomic emission spectroscopy using a Varian Liberty Series II spectroscope fitted with an axial torch (Varian, Mulgrave, Australia). Concentrations of progastrin were calculated from the absorbance at 280 nm, which was determined on a Cary 5 spectrophotometer (Varian, Mulgrave, Australia). Cell proliferation was measured by incorporation of bromodeoxyuridine. The conditionally transformed mouse colon cell line YAMC (16Whitehead R.H. VanEeden P.E. Noble M.D. Ataliotis P. Jat P.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 587-591Crossref PubMed Scopus (339) Google Scholar) was plated onto sterile 14-mm coverslips in 24-well plates at a density of 25,000 cells/well in RPMI 1640 medium containing 5% fetal calf serum and 1 unit/ml γ-interferon and grown overnight at 33 °C. The cells were then transferred to 39 °C and washed once in PBS, and the medium was replaced with RPMI 1640 containing l-glutamine but without fetal calf serum or interferon. After 24 h cells were incubated for a further 18 h in the same medium containing 1% fetal calf serum and 100 μm bromodeoxyuridine, with or without the factors to be tested. The cells were then rinsed once with PBS, fixed for 5 min in ice-cold methanol at 4 °C, and washed three times in PBS. The cells were permeabilized in PBS containing 0.5% HCl for 10 min, washed once in PBS, and incubated for 1 h with a mouse anti-bromodeoxyuridine antibody. After three rinses in PBS, the cells were incubated with FITC-labeled goat anti-mouse IgG for 30 min. After three further rinses the coverslips were mounted on slides with cytifluor and observed on a fluorescence microscope. COS7 cells were transiently transfected by the DEAE-dextran method as described previously (21Mantamadiotis T. Baldwin G.S. Biochem. Biophys. Res. Commun. 1994; 201: 1382-1389Crossref PubMed Scopus (31) Google Scholar). One day before transfection, 0.7–1.0 × 106 COS cells were seeded in 10-cm plates in Dulbecco's modified Eagle's medium and grown in 5% CO2 such that on the day of the cells were the day of transfection, a solution was prepared by of 0.5 ml 2 DEAE-dextran in PBS to 0.5 ml of in PBS containing 3.5 μg/ml plasmid encoding either the human CCK-A or the human receptor K. C. M. P. Biochem. Biophys. Res. Commun. 1995; PubMed Scopus Google Scholar). The medium was and the cells were washed once with PBS and gently at 37 °C for 20 min in the The solution was then replaced with 10 ml of 100 μm and the cells were incubated at 37 °C for 3.5 h. After the solution was and the cells were washed with Dulbecco's modified Eagle's medium and grown in Dulbecco's modified Eagle's medium with 10% fetal calf serum the the transfected cells were with onto a 24-well and grown for a further h prior to the receptor binding Binding of progastrin to either the human CCK-A receptor or the human gastrin/CCK-B receptor was measured by for and binding as described by and coworkers M. M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). COS7 cells were grown to as described washed once with PBS, and then incubated for min at 37 °C in 150 of Dulbecco's modified Eagle's medium 150 μm and were then washed with PBS and with of 1 m were in a at of and of the bound in the of were fitted as previously described F. Baldwin G.S. Cancer 1998; PubMed Scopus Google Scholar). are expressed as the ± except and were analyzed by of and by of on was a in the or of each the were with the by or of were Human progastrin was expressed in E. coli as a fusion protein with glutathione S-transferase The fusion protein was isolated by binding to glutathione-agarose beads to Frangioni and Neel (19Frangioni J.V. Neel B.G. Anal. Biochem. 1993; 210: 179-187Crossref PubMed Scopus (833) Google Scholar). human progastrin was cleaved from the fusion protein bound to glutathione-agarose beads by treatment with thrombin human progastrin was purified by reverse phase HPLC (Fig. The absorbance peak at well with the peak of observed with antiserum which was raised against an undecapeptide consisting of the C-terminal decapeptide of progastrin with an additional tyrosine residue at the N terminus for iodination (9Ciccotosto G.D. McLeish A. Hardy K.J. Shulkes A. Gastroenterology. 1995; 109: 1142-1153Abstract Full Text PDF PubMed Scopus (183) Google Scholar). The that the recombinant human progastrin the terminus of progastrin was confirmed by ionization mass spectrometry. The molecular mass of recombinant human progastrin was ± which is in with the mass of predicted for human The N-terminal amino acid sequence of recombinant human progastrin determined by Edman was which to of human We conclude that the recombinant human progastrin of of human the N-terminal sequence of human progastrin is appears that thrombin has cleaved the peptide the residue at 5 and the residue at Previous have that human [Nle11]-gastrin13 and [Nle15]-gastrin17 bind three Ca2+ ions in trifluoroethanol (12Peggion E. Mammi S. Palumbo M. Moroder L Wunsch E. Biopolymers. 1983; 22: 2443-2457Crossref PubMed Scopus (25) Google Scholar, 13Peggion E. Mammi S. Palumbo M. Moroder L Wunsch E. Biopolymers. 1984; 23: 1225-1240Crossref PubMed Scopus (15) Google Scholar). The presence of metal ions in recombinant human progastrin6–80 was by inductively coupled plasma atomic emission spectroscopy. The revealed that recombinant human progastrin6–80 ± calcium ± = other were detected The calcium ion was not by dialysis at pH against 10 μm EDTA (Fig. or 100 μm EDTA not by treatment with urea containing 10 mm EDTA (Fig. or by dialysis at pH against 100 μm EDTA not human progastrin6–80 stimulated proliferation of the conditionally transformed mouse colon cell line YAMC in a with effects in the range 10 to 100 pm The of recombinant human progastrin6–80 on YAMC cells was by either the CCK-A or the gastrin/CCK-B at concentrations as high as 10 μm (Fig. 5 Gastrin17gly stimulated proliferation of YAMC cells in the range 1 pm to 1 nm, as has been reported previously with a F. Imdahl A. Mantamadiotis T. Ciccotosto G.D. Shulkes A. Baldwin G.S. Gastroenterology. 1997; 113: 1576-1588Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). of progastrin binding were to CCK-A and gastrin/CCK-B receptors, which have been characterized at the nucleotide sequence Binding of recombinant human progastrin6–80 to either the human CCK-A or human gastrin/CCK-B receptor was by for the binding of to transiently transfected COS cells as described human progastrin6–80 had no on the binding to the gastrin/CCK-B receptor at concentrations as high as 100 nm and stimulated the binding of to the CCK-A receptor. and gastrin17 were used as for of binding to the CCK-A and gastrin/CCK-B In the of progastrin of from specific binding sites on COS cells transfected with encoding either the CCK-A receptor or the gastrin/CCK-B receptor was observed in the presence of or The determined by of the to a single model were ± 5 nm for the CCK-A receptor and ± 15 nm for the gastrin/CCK-B receptor. are than previously reported for the affinities of the human CCK-A receptor nm for A. K. Biochem. Biophys. Res. Commun. 1993; PubMed Scopus Google and human gastrin/CCK-B receptor nm for gastrin17 A. K. Biochem. Biophys. Res. Commun. PubMed Scopus Google for The may be in to the presence of than of binding sites K. C. M. P. Biochem. Biophys. Res. Commun. 1995; PubMed Scopus Google Scholar). In this the and purification of recombinant human progastrin6–80 from E. coli is reported for the first Progastrin was synthesized as a fusion protein with (Fig. and purified by the affinity of for glutathione-agarose (Fig. After treatment of the bound fusion protein with progastrin6–80 was into the which was separated from the by centrifugation. purification of the supernatant by reverse phase HPLC in of recombinant human progastrin that were by gel electrophoresis (Fig. and mass spectrometry. The molecular mass of recombinant human progastrin determined by ionization mass spectrometry was ± in with the of for human peptide be by cleavage and in the progastrin sequence which is with the thrombin sequence and are amino and and are amino acids Eur. J. Biochem. 1985; PubMed Scopus Google Scholar). of from a human by and R. K. J.E. P. T. J. Full Text PDF PubMed Google Scholar) had previously revealed that the signal peptide of was amino acids and that an additional cleavage and Rehfeld and Johnsen (10Rehfeld J.F. Johnsen A.H. Eur. J. Biochem. 1994; 223: 765-773Crossref PubMed Scopus (50) Google Scholar) purified and from human