Biochimica et Biophysica Acta, 1039 (1990) 135-141

135

Elsevier BBAPRO 33685

Purification and characterization of recombinant human activin B Charles H. Schmelzer 1, Louis E. Burton 1, Cathleen M. Tamony 1, Ralph H. Schwall 2, Anthony J. Mason 3 and Nanette Liegeois 1 t Recooery Process R & D, 2 Pharmacological Sciences and 3 Deoelopmental Biology, Genentech, lnc., San Francisco, CA (U.S.A.)

(Received11 September1989) (Revised manuscript received2 February 1990)

Key words: Activin B; ActivinA; Protein purification; Growth factor Recombinant human activin B has been isolated to more than 95% purity from a mammalian kidney cell line. Activin B is a covalently-linked homodimer with an apparent molecular mass of 25.9 kDa (unreduced) and 15.2 kDa (reduced) as determined by SDS-polyacrylamide-gel electrophoresis. On gel filtration in 6 M guanidine hydrochloride, activin B chromatographs with an apparent molecular mass of 11 kDa, whether reduced or not. The amino-terminal sequence of the purified protein is consistent with the expected sequence derived from the fl subunit of inhibin B. The amino acid composition of the purified molecule agrees with the expected theoretical composition of the fl subunit of inhibin B. Activin B has an apparent pl of 4.6 as determined by isoelectric focusing in 6 M urea and 4.7 as determined by chromatofocusing in 6 M urea. The extinction coefficient is 1.8.

Introduction Activin is a member of the transforming growth factor-fl (TGF-fl) gene family [1,2]. Biologically, activin has been shown to stimulate both the secretion of follicle stimulating hormone (FSH) from anterior pituitary cells and the accumulation of hemoglobin in the K562 erythroid leukemic cell line [1,3]. Inhibin, another member of the TGF-fl gene family, shows biological effects opposite to those exhibited by activin in the systems described above [1,4]. Both inhibin [1,5,6] and activin [7,8] have been isolated from natural sources. Activin isolated from natural material is a covalent dimer exhibiting two forms, flAflA and flAflB" On the other hand, inhibin exhibits two covalent heterodimeric forms, aft A and aflB. The fl subunits are identical in

Abbreviations: TGF-fl, transforming growth factor-fl; FSH, follicle stimulating hormone; Mes, 2-(N-morpholino)ethanesulfonic acid; Mops, 3-(N-morphohno)propanesulfonicacid; SSFF, S-Sepharose Fast Flow; NMWL, nominal molecular weight limit; TFA, trifluoroacetic acid; SDS, sodium dodecyl sulfate; PAGE, polyacrylamidegel electrophoresis; Bistris, bis-(2-hydroxyethyl)iminotris(hydroxymethyl)methane; RP-HPLC, reversed-phasehigh-performanceliquid chromatography; IEF, isoelectric focusing; TCF, tissue culture fluid; UF/DF, ultrafiltered/diafiltered. Correspondence: C.H. Sehraelzer,Genentech, Inc., RecoveryProcess R&D, 460 Point San Bruno Blvd., South San Francisco, CA 94080, U.S.A.

both activin and inhibin. Recombinant human activin A (flAflA) has been isolated and shown to have biochemical properties and biological activities similar to its native counterpart isolated from ovarian follicular fluid [7-9]. To date, activin B (flBfla) has been postulated [2], but not yet isolated from natural sources. Although fib mRNA has been detected in the ovary, testis, placenta, pituitary, and whole brain [10], only recently has the fib m R N A been found alone in granulosa cells of small antral follicles [11]. Based on the nucleotide sequence of the c D N A encoding the fib subunit [12], recombinant activin B has recently been expressed in mammalian kidney cells [13]. We present here a method for the purification and some biochemical characterization of the recombinant activin B molecule purified from those cells. This is the first report describing the purification of this novel protein. The availability of purified activin B is crucial to the complete structural and functional characterization of the protein and provides an opportunity to increase our understanding of the biological role of this interesting protein. Experimental procedures Materials

S-Sepharose Fast Flow, phenyl-Sepharose CL-4B, Sephacryl S-300 HR, low-molecular-weight SDS standards, isoelectric focusing standards (3-10), PhastGel IEF 3-9, polybuffer 74, Mono P H R 5 / 2 0 and a Superose 12 H R 10/30 were purchased from Pharmacia LKB

0167-4838/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)

136 Biotechnology, Inc. YM10 ultrafiltration membranes were from Amicon. A microdialysis system was purchased from Bethesda Research Laboratories. The Vydac C a 214TP]010 reversed-phase H P L C column (1.0 z 25 cm) was obtained from P h a s e Separations Group. Pellicon cassette membranes (10000 N M W L , Cat. No. P T G C 000 05) were purchased from Millipore. Enhance was purchased from New England Nuclear. Acetonitrile was obtained from Baker Chemical Co. Trifluoroacetic acid was from Pierce. Bio-Lyte 3 / 1 0 carrier ampholytes were from Bio-Rad. Myoglobin (M9267), carbonic anhydrase (C6403), trypsinogen (Tl146), trypsin inhibitor (T1021), /3-1actoglobulin A (L5137), ovalbumin (A2512), cytochrome c (C7752), aprotinin (Al153), Blue dextran (D4772) and dithiothreitol were purchased from Sigma. Urea was from Mallinckrodt and was prepared as a stock 7.5 M solution and deionized using a mixed bed resin prior to use. All other chemicals were of reagent grade.

Methods Expression of activin B. Details of the expression of activin B are described elsewhere [13]. Briefly, a fulllength coding sequence for the fib subunit of inhibin was inserted into a cytomegalovirus expression vector which was used to transfect a mammalian kidney cell line [9,14]. Neomycin-resistant clones were selected and shown to secrete biologically competent activin B [13]. Purification of actioin B. 40 1 of tissue culture fluid (TCF) containing recombinant activin B from mammalian kidney cells were concentrated approx. 80-fold at 4 ° C and diafiltered into 25 m M Mes, 6 M urea (pH 6.5) using a pellicon tangential flow membrane (polysulfone) concentrator (10 square feet N M W L 10000). All chromatography steps were performed at room temperature unless otherwise indicated. The U F / D F concentrated T C F was applied to a 5 x 13 cm SSFF column equilibrated in 25 m M Mes, 6 M urea (pH 6.5) at a flow rate of 15 m l / m i n . The flow-through fraction, containing activin B, was adjusted to 0.8 M NaC1 and applied to a 2.5 x 9 cm phenyl Sepharose CL-4B column equilibrated in 25 m M Mops, 0.8 M NaCI, 6 M urea, (pH 7.0) at a flow rate of 5 m l / m i n . The column was washed with equilibration buffer (3 bed volumes) until the A280 returned to near baseline, followed by 9.5 bed volumes of 25 m M Mops, 6 M urea (pH 7.0). The column was eluted with a solution consisting of 40% ethanol and 60% 25 m M Mops, 6 M urea (pH 7.0). Activin B was pooled and concentrated approx. 10-fold in an Amicon stirred cell using a YM10 membrane. The concentrated pool was applied to a Sephacryl S-300 H R column (2.5 x 110 cm) equilibrated with 1 M acetic acid, 7.1 M urea at a flow rate of 0.8 m l / m i n at 4 ° C . The activin B was pooled and loaded directly onto a Vydac C 4 R P - H P L C (1.0 x 25 cm) column at 4 m l / m i n using a Waters 600 E H P L C system. Solvent A was

aqueous 0.1% trifluoroacetic acid (TFA) and solvent B was 0.1% T F A in acetonitrile. After loading of sample, the flow rate was reduced to 3 m l / m i n and the column was washed for 5 min with 32% B. Activin B was eluted using a linear gradient of 32 to 38% B in 75 min. Fractions containing activin B were pooled and stored at 4 ° C for biological and chemical characterization. SDS polyacrylamide gel electrophoresis. Slab gel electrophoresis was performed in a 12.5% resolving gel with a 4% stacking gel according to the method of Laemmli [15]. The gels were silver-stained according to Morrisey [161. Gel filtration. The molecular weight of activin B was determined by gel filtration on a Superose 12 column (1 x 30 cm) equilibrated in 25 m M Tris-HCl (pH 7.2) containing 6 M guanidine hydrochloride. The flow rate was 0.5 m l / m i n . Reduced activin samples (10 ~g) were prepared prior to chromatography by reduction at 80 o C for 5 min in equilibration buffer containing 100-fold molar excess of dithiothreitol over disulfide bonds. Other samples were dissolved in equilibration buffer at room temperature prior to chromatography. The Superose 12 column was calibrated using ovalbumin ( M r = 43 000), carbonic anhydrase ( M r = 30 000), cytochrome c ( M 12400) and aprotinin ( M r = 6500). Isoelectric focusing. The p I for activin B was determined using a Pharmacia Phast gel system (PhastGel IEF 3-9). Prior to use the I E F gel was soaked in Bio-Lyte 3 / 1 0 carrier ampholyte solution containing 6 M deionized urea. The p H gradient was determined using calibrated p I standards: (Broad p I calibration kit 3-10: myoglobin (6.76, 7.16) carbonic anhydrase (5.85), trypsin inhibitor (4.55), and trypsinogen (9.3)). Protein sequencing. The amino-terminal sequence of activin B was determined using an Applied Biosystem 477 sequenator with an on line 120A HPLC. Amino acid analysis. Amino acid compositions were determined using a Beckman 6300 amino acid analyzer. Samples were hydrolyzed for 24 h at l l 0 ° C in 6 M HC1 prior to analysis. Chromatofocusing. Chromatofocusing was performed using a Mono P H R 5 / 2 0 column. The column was equilibrated in 25 m M Bistris (pH 6) containing 6 M urea. The flow rate was 0.5 m l / m i n . Purified activin B was eluted from the Mono P column using 10% polybuffer 74 (pH 4.0) in 6 M urea. 1-ml fractions were collected. The p H gradient profile was determined using a Corning 125 p H meter. Extinction coefficient. The extinction coefficient for activin B was determined using a Hewlett Packard 8452A Diode Array spectrophotometer. The protein was dialyzed and the absorbance was determined in 0.05 M acetic acid. The concentration of activin B was determined by amino acid analysis. Protein assay. Protein concentration was determined by the method of Bradford [17] using the Pierce protein r

=

137 assay reagent. Activin B quantitated by amino acid analysis was used as the standard. Protein concentrations of purified samples were also determined either by amino acid analysis or by absorbance at 280 nm using 280nm of 1.8. 80.1% I cm Immunoprecipitations. Transfected or stable clones expressing either activin A or activin B were grown in the presence of [35S]methionine and [35S]cysteine [13]. For immunoprecipitation of activin A, protein A-purified monoclonal antibody prepared against recombinant activin A was used. For activin B, ammonium sulfateprecipitated goat polyclonal antibody raised against purified recombinant activin B was used. Recombinant activin A was immunoprecipitated from the labelled supernatants (50/~ 1) with 2/tg of affinity-purified activin A IgG at room temperature for 1 h. Recombinant activin B was immunoprecipitated from labeled supernatants (50 #1) with 2 #g of activin B IgG at room temperature for 1 h. Immune complexes were precipitated with protein A. Samples were separated on a 12% SDS-polyacrylamide gel, fixed in acetic acid, treated with Enhance, dried and analyzed by autoradiography. Bioassay. Activin B concentrations were determined by the stimulation of FSH release in cultured pituitary cells as previously described [9]. Activity is expressed as the concentration of activin B required to give a halfmaximal response in the assay (EDs0). 1 unit of activity is equivalent to 2.2 ng/ml of purified activin B. EDs0 values were calculated using a four-parameter curve-fitting program [18]. Results

Purification We describe here a simple, four-step purification which yields highly purified (more than 95%) activin B. The results of cation exchange chromatography of the concentrated tissue culture fluid on S-Sepharose fast flow are shown in Fig. 1. Activin B eluted in the flow

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Purification and characterization of recombinant human activin B.

Recombinant human activin B has been isolated to more than 95% purity from a mammalian kidney cell line. Activin B is a covalently-linked homodimer wi...
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