1 Field of Microbiology, Cornell University, Ithaca, NY 14853, USA 2 Boyce Thompson Institute, Cornell University, Ithaca, NY 14853, USA 3 Department of Food Science, Cornell University, Ithaca, NY 14853, USA
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Abstract |
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Keywords: characterization/expression/glycosylation/ß-lacto-globulin/Pichia pastoris
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Introduction |
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BLG shows strong sequence homology to the human protein glycodelin [also known as placental protein-14 or PP14 (Huhtala et al., 1987; Julkunen et al., 1988
)]. Glycodelin is a glycoprotein with a molecular weight of 28 kDa and is found in several tissues including the decidualized endometrium, seminal plasma, hematopoietic tissues of the bone marrow and glandular epithelia (Julkunen et al., 1988
; Morrow et al., 1994
; Morris et al., 1996
, Kamarainen et al., 1997
). This protein exhibits several interesting biological functions including inhibition of lymphocyte proliferation and cytokine secretion by lymphocytes (Bolton et al., 1987
; Pockley et al., 1988
), inhibition of sperm-egg binding (Oehninger et al., 1995
) and stimulation of cellular differentiation (Kamarainen et al., 1997
). One isoform of this protein referred to as glycodelin-A (GdA), isolated from the decidualized endometrium, was found to inhibit sperm-egg binding (Oehninger et al., 1995
) in a hemizona assay. Another isoform of glycodelin termed glycodelin-S (GdS), found in the seminal plasma, did not exhibit this property. This difference in properties has been attributed to differences in their glycosylation pattern (Morris et al., 1996
).
Glycodelin has three sites for glycosylation (Huhtala et al., 1987; Julkunen et al., 1988
) with a consensus sequence of NXT/S, where X can be any amino acid residue other than proline. An alignment of glycodelin and BLG revealed the glycosylation sites to correspond to the residues 6365 (S1), 8587 (S2) and 2830 (S3) on BLG (Figure 1
). Common variants of wild-type BLG, namely the A and B variants, do not have any consensus sites for glycosylation (Jamieson et al., 1987
). However, Bell et al. (1970) reported a rare BLG variant called ß-lactoglobulin droughtmaster (BLGDr) which carries a single carbohydrate chain.
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Even though differential glycosylation of glycodelin is considered to be responsible for the variety of biological functions it displays, it is not known which specific glycosylation pattern is necessary for a particular function. A number of human glycoproteins expressed in P.pastoris have been shown to have retained their native structure and biological properties (Caputo et al., 1999; Gupta and Dighe, 1999
; Lerner et al., 1999
).
In this study, the glycosylation sites found in glycodelin were introduced into BLG by site-directed mutagenesis with the aim of developing BLG as a molecular mimic of glycodelin. The resulting mutant proteins were overexpressed in P.pastoris and their glycosylation profile was characterized by a number of physical methods.
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Materials and methods |
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Escherichia colii TG1 (K12 (lac-pro), supE, thi, hsd
5/F'[traD36, proAB, lacIq, lacZ
M15]) was used as the host strain to construct vectors for the expression of the BLG variants. The expression vector used was pPIC9 (Invitrogen, Carlsbad, CA). It carries the alcohol oxidase I promoter (AOX I), the
mating factor signal sequence from S.cerevisiae and the His4 selectable marker. Plasmid BLG/pPIC9, prepared from E.coli TG1 (Kim et al., 1997
), was used as the source of wild-type BLG gene for site-directed mutagenesis. P.pastoris GS115 (his4) was used as the host strain for protein expression (Cregg et al., 1985
). All media used for E.coli and P.pastoris were prepared as described by Kim et al., (1997).
DNA manipulation
Plasmid DNA was purified using the Qiagen plasmid preparation kit (Qiagen, Santa Clarita, CA). Restriction enzymes were purchased from New England Biolabs (Beverly, MA) and DNA ligase from GIBCO (Gaithersburg, MD). Purification and ligation of digested plasmid DNA or PCR products were performed according to published protocols (Ausubel et al., 1990).
Mutagenesis
The megaprimer method of site directed mutagenesis (Sarkar and Sommer, 1990) was used to introduce mutations. This method makes use of PCR for the same purpose. The mutagenic primers used are shown in Table I
. The flanking primers used were 5' XhoI and 3' EcoRI (Table I
) (Kim et al., 1997
). PCR-I was prepared as follows: 100 ng template, 1 µl mutagenic primer (50 pmol), 1 µl 5' or 3' flanking primer (50 pmol), 12 µl magnesium chloride (25 mM), 10 µl reaction buffer (10x), 2.5 µl deoxynucleotides (2.5 mM), 0.5 µl Taq DNA polymerase (5 units/µl) (Perkin-Elmer Cetus, Norwalk, CT) in a final volume of 100 µl with D2O. This mixture was subjected to 1 cycle of 95°C for 5 min, 30 cycles of 95°C for 1 min (denaturation), 55°C for 1 min (annealing), 70°C for 1 min (extension) and 1 cycle of 70°C for 15 min using a 2400 DNA Thermal Cycler (Perkin-Elmer Cetus). The PCR-I product (megaprimer) was purified using the Qiagen PCR purification kit. PCR-II was set up in the same way as PCR-I except that in this case 2 µl of the megaprimer and 1 µl of the other flanking primer (50 pmol) were used. This mixture was subjected to 1 cycle of 94°C for 5 min, 30 cycles of 94°C for 1 min (denaturation), 60°C for 1 min (annealing), 72°C for 1 min (extension) and 1 cycle of 72°C for 15 min. The PCR-II product was purified by gel extraction using the Qiagen gel extraction kit. The purified PCR-II product was digested using XhoI and EcoRI and then cloned into the XhoI and EcoRI sites of pPIC9. The BLG portion of the expression cassette was sequenced using 5' XhoI and 3' EcoRI primers to check if the mutations had been introduced correctly.
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Transformation of P.pastoris and screening for integrated vector, methanol utilization (Mut) phenotype and protein expression (test-tube cultures) were done according to Kim et al., (1997).
To express BLG in P.pastoris, a single colony of S2, S12 and S123 strains of P.pastoris was grown separately in 25 ml of BMGY media, overnight at 30°C with continuous shaking at 250 r.p.m. A 2 l baffle flask containing 500 ml of BMGY was inoculated with the overnight culture and grown for 2 days at 30°C at 250 r.p.m. After 2 days, the cells were collected by centrifugation at 2500 g. The cells were resuspended in 500 ml of BMMY and induced by adding 0.5% methanol every 24h. Protein expression was checked every 24h by SDSPAGE. After 4 days of induction, the culture was centrifuged at 2500 g as before and the supernatant was collected.
Protein purification
The cell-free supernatant was subjected to two rounds of cation-exchange chromatography on a 5 ml Econo S cartridge (Bio-Rad Laboratories, Richmond, CA) using the Biologic HR system (Bio-Rad Laboratories), according to Denton et al. (1998). The pooled fractions were concentrated to 5 ml by ultrafiltration using a YM-10 membrane (Millipore, Bedford, MA). The retentate was further concentrated to 200 µl using Centricon-10 concentrators (Millipore). The yield was determined in the case of each variant by SDSPAGE.
SDSPAGE
Cell-free supernatant and purified fractions from S2, S12 and S123 cultures were analyzed using 12.5% SDSPAGE (Laemmli) under denaturing conditions. Wild-type BLG (Sigma, St. Louis, MO) was used as the protein standard. The proteins were stained with Coomassie Brilliant Blue dye (Bio-Rad Laboratories).
Western blotting
Supernatants from S2, S12 and S123 cultures were run on denaturing SDSpolyacrylamide gels and then electroblotted to a nitrocellulose membrane (Micron Separation, Westboro, MA). A rabbit polyclonal antibody against BLG (Batt et al., 1990) was used as the primary antibody. The primary antibodyantigen complexes were detected using anti-goat anti-rabbit antiserum conjugated to horseradish peroxidase (HRP) and developed using a Bio-Rad HRP substrate.
Mass spectrometry
Purified S2, S12 and S123 proteins were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) using a Lasermat 2000 (Thermo-bioanalysis, Franklin, MA). The matrix used was 3,5-dimethoxy-4-hydroxycinnamic acid and the accelerating voltage was ~20 000 V. Purified protein samples were dissolved in a mixture of 70% acetonitrile and 0.1% trifluoroacetic acid at a concentration of 10 µM. Approximately 1 µl of the sample was resuspended in 1 µl of the matrix solution and applied to the matrix. The mass spectrometry protocol followed was according to the manufacturer's instructions.
Glycosylation profiling
For glycosylation profiling, 200 µg of purified S2, S12 and S123 proteins were used. The proteins were first digested with PNGaseF (Boehringer Mannheim, Indianapolis, IN) and then labeled with 2-aminobenzamide dye (2-AB) (Oxford Glycosystems, Oxford, UK). Oligosaccharide digestion, labeling and HPLC were performed according to Joshi et al. (2001).
Characterization of 2AB-labeled oligosaccharides by HPLC was performed on a Glycosep-N column (Oxford Glycosystems) using a Model 2690 HPLC system (Waters, Milford, MA) and a Waters Model 474 fluorometric detector. A partial hydrolyzate of dextran that produced peaks composed of 123 glucose units was used as the reference to calibrate the column. A calibration curve of the HPLC glucose unit values was constructed using the elution positions of the dextran peaks (Guile et al., 1996).
Exoglycosidase digestions using jack bean -mannosidase (Oxford Glycosystems, Rosedale, NY), Aspergillus saitoi (
-1,2)-mannosidase (Oxford Glycosystems) and recombinant Xanthomonas manihotis (
-1,6) mannosidase (New England Biolabs) were performed according to the manufacturer's instructions using 10 µl of 2-AB-labeled glycans for each reaction. Endoglycosidase digestion using endoglycosidase H (New England Biolabs) was also performed according to the manufacturer's instructions. The glycosylation profile of each protein was determined by HPLC.
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Results |
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In order to express the BLG variants in P.pastoris, the mutant genes were cloned into the multiple cloning site of the Pichia expression vector pPIC9 using the restriction sites XhoI and EcoRI. This resulted in a fusion between the -mating factor signal sequence and the BLG gene at the 5' end. The first expression cassette to be constructed was S2-BLG/pPIC9. This construct carried the BLG mutant, in which the glycosylation site S2 had been introduced by site-directed mutagenesis. The mutations at this site were D85N, A86Y and L87T. This construct was used as the template to introduce the glycosylation site S1 generating the variant S12. The construct S12-BLG/pPIC9 carried the mutations G64N and E65S in addition to the three mutations corresponding to S2. The S12-BLG/pPIC9 construct was then used as the template to construct S123-BLG/pPIC9. The additional mutation corresponding to the third glycosylation site was D28N. The BLG portion of the expression cassettes was sequenced to determine if the mutations had been introduced correctly.
Protein expression in P.pastoris
The mutated BLG genes cloned into pPIC9 were electroporated into P.pastoris and His+ transformants were selected. Twelve His+ colonies were chosen randomly to screen for BLG insert using XhoI and EcoRI primers. Colonies that carried the BLG gene were screened for expression and methanol utilization (Mut) phenotype. A single Mut+ colony of each variant was selected for the large-scale expression of protein in shake flasks. After growth on glycerol for 2 days and induction by methanol for 4 days, the cell-free supernatant from the shake flask cultures was collected. SDSPAGE analysis of the supernatants revealed a single 18 kDa band in the case of the S2 mutant. In addition to the 18kDa band, a heterogeneous population of proteins was observed in the case of S12 and S123, with molecular weight ranging from ~21 to ~30 kDa (Figure 2A).
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Protein purification and yield
The cell-free supernatant from shake flask cultures of P.pastoris was purified by cation-exchange chromatography. Fractions containing the protein were pooled and concentrated. The amount of protein obtained in the case of each variant was estimated by SDSPAGE to be ~100 mg/l.
Endoglycosidase H treatment
To confirm the glycosylation of BLG, all three mutant proteins were digested with endoglycosidase H, which specifically cleaves the high-mannose core of N-linked glycoproteins (Figure 3). Upon SDSPAGE analysis, no shift in migration was observed in the case of S2, indicating no glycosylation. However, on endoglycosidase H treatment of S12 and S123, the heterogeneous population of proteins disappeared, resulting in a single band corresponding to 18 kDa. This showed that the heterogeneity in molecular weight observed in the case of S12 and S123 proteins was due to differences in glycosylation of BLG.
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Purified S2, S12 and S123 proteins were analyzed by MALDI-MS in order to determine accurately the molecular weight of the mutant proteins. The major peak in the case of S2, S12 and S123 was at 18 219, 20 997 and 22 829 Da, respectively (data not shown). The molecular weight of wild-type BLG expressed in P.pastoris was determined to be 18 676 Da (Kim et al., 1997). This corresponded to BLG carrying four additional amino acids (GluAlaGluAla) at the N-terminus due to incomplete signal sequence processing. Taking this to be the molecular weight of unglycosylated BLG, the number of hexose residues in S12 and S123 was estimated to be 12.9 and 23.1, respectively. In the case of S2 a secondary peak was observed at 18 614 Da in addition to the peak at 18 219 Da. N-Terminal sequencing of S2 revealed heterogeneity in signal sequence processing. The predominant species was the fully processed protein which did not carry the GluAla repeats. A smaller population carrying the GluAlaGluAla sequence was also observed. This showed that the two peaks observed in the case of S2 were due to differences in signal sequence processing rather than differences in glycosylation. The molecular weights obtained for S12 and S123 proteins correspond to the major species within the heterogeneous glycosylated population as observed by SDSPAGE analysis.
Glycosylation profiling
The HPLC profile of N-linked glycans released from the three variants by PNGaseF treatment revealed a number peaks (Figure 4). The number of glucose units contained in each peak was estimated by comparison of their retention time with that of the dextran standard used to calibrate the column. In order to estimate the polymerization state of the glycans, it was assumed that the entire pool comprised N-linked high-mannose structures, as shown by previous studies on glycosylation in P.pastoris (Cregg and Higgins, 1995
; Miele et al., 1997
). The number of glucose units corresponding to each peak was used to determine the polymerization state of the glycans. A value of 0.9 glucose units was assigned for mannose and 0.5 glucose units for N-acetylglucosamine, based on the results of Guile et al. (1996). From these calculations, the range of high-mannose structures in the total glycan pool was determined to be Man915GN2 in the BLG variants (Figure 4AC
). Digestion of glycans from all three variants with jack bean mannosidase (Oxford Glycosystems), which cleaves all
-mannose linkages, removed all peaks except Man1GN2 (Figure 4D
). This confirmed that the glycosylation was of the N-linked high-mannose kind typically seen in P.pastoris.
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In order to determine the number of outer -1,2 mannose linkages, the glycans from S123 were digested with
-1,2 mannosidase. The peak that remained after digestion with this enzyme was Man6GN2 (Figure 4E
). If all the outer chains were composed of
-1,2 mannose linkages, then the product of
-1,2 mannosidase digestion would be a Man5GN2 peak. Therefore, the Man6GN2 peak obtained after
-1,2 mannosidase digestion suggested the presence of a single
-1,6 linkage within the core Man8GN2 structure. From this, it was deduced that the Man914GN2 peaks observed in the case of S123 protein were obtained by the extension of the Man6GN2 structure by a minimum of three to a maximum of eight
-1,2-linked mannose residues (Figure 5
). Digestion of glycans from S123 with
-1,6-mannosidase (New England Biolabs) removed all peaks except Man1GN2 (not shown). This non-specific
-mannosidase activity might be due to contaminating
-1,2- and
-1,3-mannosidase activities in the reaction.
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Discussion |
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Previously there have been studies on the non-enzymatic synthesis of glycosylated derivatives of BLG by the Maillard reaction (Burr et al., 1996; Leonil et al., 1997
). This involves the covalent addition of lactose to lysine residues of BLG during heat treatment of milk. Lysine-47 has been identified as one of the sites for lactosylation of BLG. (Leonil et al., 1997
). Subsequently, lysine-100 was also found to be a site for lactosylation of BLG (Fogliano et al., 1998
). The Maillard reaction is of significance commercially, as it causes browning of milk products upon heating. Waniska and Kinsella (1984) and Shida et al. (1994) reported the synthesis of maltosyl-, glucosaminyl- and lactosyl-BLG by chemical methods. These derivatives were formed by the reaction between the sugar residues and the reactive groups such as carboxyl, hydroxyl, sulfhydryl and amino found on the protein. A maximum of 64 glucose residues in the case of maltosyl-BLG and 16.6 glucose residues in the case of glucosaminyl-BLG were added per protein molecule. The glycosylated derivatives of BLG were found to exhibit enhanced foaming and emulsifying properties. (Waniska and Kinsella, 1984
) The structure of these oligosaccharide linkages is very different from those observed in the case of N-linked glycans. N-Linked glycans are always attached to the asparagine residue in the consensus sites for glycosylation AsnXThr/Ser (NXT/S) (Burda and Aebi, 1999
). The core structure, Man8GN2, is conserved in all eukaryotic glycoproteins. However, the composition of the outer chains varies with the organism.
Common variants of BLG, namely BLG-A and -B, are not glycosylated in the native state. However, a rare variant of BLG called ß-lactoglobulin droughtmaster (BLGDr) appears to carry a single N-linked glycosylation site (Bell et al., 1970). This variant was isolated from ADr and BDr heterozygotes and was identified to be identical in both heterozygotes. Unlike the A and B variants, the BLGDr variant did not undergo oligomerization at pH 4.7 and low temperatures. The antigenicity of the Dr variant also appeared to be different from the other two variants as it was not possible to raise antibodies against BLGDr.
In this study, physical characterization of the glycosylated BLG variants by means of SDSPAGE, Western blot analysis, endoglycosidase H treatment, mass spectrometry and glycosylation profiling confirmed the nature and extent of glycosylation. The glycosylation observed was of the N-linked high-mannose type, typically seen in P.pastoris (Montesino et al., 1998). A glycan structure accounting for the results obtained is shown in Figure 5
. Extension of the core Man8GN2 was mainly by
-1,2 linkages. The results obtained also suggest the presence of a single
-1,6 linkage within the core. This is consistent with previous glycosylation studies on P.pastoris (Cregg and Higgins, 1995
; Miele et al., 1997
). Glycosylation profiling also revealed that the BLG variants were not hyperglycosylated. The N-linked oligosaccharides observed had only 915 mannose residues as opposed to >50 residues usually observed in the case of hyperglycosylation by S.cerevisiae (Romanos et al., 1992
) and sometimes in the case of P.pastoris (Scorer et al., 1993
). The results obtained did not indicate extension of the core by
-1,3 linkages, a feature commonly seen in the case of glycosylation by S.cerevisiae.
The glycan structures obtained in this study appear to be similar in composition to those that occur at Asn28 of glycodelin-S (Morris et al., 1996). However, the outer mannose chains in the BLG variants appear to be longer than those found in GdS. The predominant carbohydrate structures found in the BLG variants were Man915GN2, whereas in the case of GdS the predominant forms observed at Asn-28 were Man57GN2.
The extent of glycosylation varied among the three mutants. The mutants with a single glycosylation site (S2) showed no glycosylation, whereas the mutant with three glycosylation sites (S123) showed the most. The mutant proteins also showed variations in the relative distribution of the different glycoforms. This might be due to local conformational characteristics of the nascent polypeptide chain resulting in variations in glycosylation processes such as core formation and core extension. Such variations could lead to the assembly of oligosaccharide chains of different lengths at the glycosylation sites. Absence of glycosylation in the S2 mutant is interesting since the corresponding glycosylation site in glycodelin, Asn85, is also not glycosylated. According to Kasturi et al. (1997), at any given AsnXSer/Thr sequence, the amino acid at the X position and the hydroxylamino acid (Ser or Thr) are important determinants of N-linked core glycosylation. The sequence at S2 is AsnTyrThr, whereas the sequences at S1 and S3 are AsnIleSer and AsnAsnSer, respectively. None of these three sequences have been mentioned as poor substrates for glycosylation. Therefore, it is likely that an inherent trait in the nascent polypeptide prevents glycosylation both at the S2 site in BLG and at the corresponding site, Asn85, in glycodelin. In this context, it is interesting that the S2 site lies on the EF loop which takes part in the pH-dependent Tanford transition of BLG (Qin et al., 1998). At low pH this loop blocks the entrance of the hydrophobic pocket of BLG and at high pH it folds open. It is possible that the mutations that comprise the S2 site in BLG affect its flexibility and permanently lock it in the closed position, which is inaccessible to glycosyl transferases. The S1 and S3 sites lie on loops CD and AB, respectively, and these loops are solvent exposed and flexible.
Convergence of function between BLG and glycodelin due to chemical modification has been observed with respect to anti-HIV activity. Wild-type BLG exhibited anti-HIV activity when modified with hydroxyphthalic anhydride (Neurath et al., 1996). A similar property was exhibited by glycodelin, when chemically modified by the same reagent (Seppala et al., 1997
). Antibodies raised against BLG have been shown to cross-react with native glycodelin (Reddy et al., 1992
) and also with recombinant glycodelin expressed in E.coli and P.pastoris (Dutta et al., 1998
). It will be interesting to determine whether the introduction of the glycosylation sites of glycodelin into BLG would confer biological functions such as immunomodulation, contraception and induction of cellular differentiation to the glycosylated BLG variants. By characterizing the glycosylation profile of these variants, it would be possible to correlate structure with biological activity of glycosylated BLG.
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Notes |
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References |
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---|
Banaszak,L., Winter,N., Xu,Z., Bernlohr,D.A., Cowan,S. and Jones,T.A. (1994) Adv. Protein Chem., 45, 89151.[ISI][Medline]
Batt,C.A., Rabson,L.D., Wong,D.W.S. and Kinsella,J.E. (1990) Agric. Biol. Chem., 54, 949956.[ISI][Medline]
Bell,K., McKenzie,H.A., Murphy,W.H. and Shaw,D.C. (1970) Biochim. Biophys. Acta, 214, 427436.[ISI][Medline]
Bolton,A.E., Pockley,A.G., Clough,K.J., Mowles,E.A., Stoker,R.J., Westwood,O.M.R. and Chapman,M.G. (1987) Lancet, i, 593595.
Burda,P. and Aebi,M. (1999) Biochim. Biophys. Acta 1426, 239257.[ISI][Medline]
Burr,R., Moore,C.H. and Hill,J.P. (1996) Milchwissenschaft, 51, 488492.[ISI]
Caputo,E., Carratore,V., Ciullo,M., Tiberio,C., Mani,J.C., Piatier,T.D. and Guardiola,J. (1999) Eur. J. Biochem., 265, 664670.
Cregg,J.M. and Higgins,D.R. (1995) Can. J. Bot., 73, S891S897.[ISI]
Cregg,J.M., Barringer,K.J., Hessler,A.Y. and Madden,K.R. (1985) Mol. Cell. Biol., 5, 33763385.[ISI][Medline]
Dell,A., Morris,H.R., Easton,R.L., Panico,M., Patankar,M., Oehninger,S., Koistinen,R., Koistinen,H., Seppala,M. and Clark,G.F. (1995) J. Biol. Chem., 270, 2411624126.
Denton,H., Smith,M., Husi,H., Uhrin, D., Barlow,P.N., Batt,C.A. and Sawyer,L. (1998) Protein Express. Purif., 14, 97103.[ISI][Medline]
Dutta,B., Mukhopadhyay,D., Roy. N., Das,G. and Karande,A.A. (1998) Protein Express. Purif., 14, 327334.[ISI][Medline]
Flower,D.R., North,A.C.T. and Attwood,T.K. (1993) Protein Sci., 2, 5361.
Fogliano,V., Monti,S.M., Visconti,A., Randazzo,G., Facchiano,A.M., Colonna,G. and Ritieni,A. (1998) Biochim. Biophys. Acta, 1388, 295304.[ISI][Medline]
Futterman,S. and Heller,J. (1972) J. Biol. Chem., 247, 51685172.
Grinna,L.S. and Tschopp,J.F. (1989) Yeast, 5, 107116.[ISI][Medline]
Guile,G.R., Rudd,P.M., Wing,D.R., Prime,S.B. and Dwek,R.A. (1996) Anal. Biochem., 240, 210226.[ISI][Medline]
Gupta,C.S. and Dighe,R.R. (1999) J. Mol. Endocrinol., 22, 273283.
Huhtala,M.L., Seppala,M., Narvanen,A., Palomaki,P., Julkunen,M. and Bohn,H. (1987) Endocrinology, 120, 26202622.[Abstract]
Jamieson,A.C., Vandeyar,M.A., Kang,Y.C., Kinsella,J.E. and Batt,C.A. (1987) Gene, 61, 8590.[ISI][Medline]
Joshi,L. Davis,T.R., Mattu,T.S., Rudd,P.M., Dwek,R.A., Shuler,M.L. and Wood,H.A. (2001) Biotechnol. Prog., in press.
Julkunen,M., Seppala,M. and Janne,O.A. (1988) Proc. Natl Acad. Sci. USA, 85, 88458849.[Abstract]
Kamarainen,M., Seppala,M., Virtanen,I. and Andersson,L.C. (1997) Lab Invest., 77, 565573.[ISI][Medline]
Kasturi,L., Chen,H. and Shakin,E.S.H. (1997) Biochem. J., 323, 415419.[ISI][Medline]
Kim,T., Goto,Y., Hirota,N., Kuwata,K., Denton,H., Wu,S.Y., Sawyer. L. and Batt,C.A. (1997) Protein Eng., 10, 13391345.[Abstract]
Leonil,J., Molle,D., Fauquant,J., Maubois,J.L., Pearce,R.J. and Bouhallab,S. (1997) J. Dairy Sci., 80, 22702281.
Lerner,M.N., Gimi,K., Urbatsch,I.L., Gros,P. and Senior,A.E. (1999) J. Biol. Chem., 274, 3471134718.
Miele,R.G., Nilsen,S.L., Brito,T., Bretthauer,R.K. and Castellino,F.J. (1997) Biotechnol. Appl. Biochem., 25, 151157.[ISI][Medline]
Montesino,R., Garcia,R., Quintero,O. and Cremata,J.A. (1998) Protein Express. Purif., 14, 197207.[ISI][Medline]
Morris,H.R., Dell,A., Easton,R.L., Panico,M., Koistinen,H., Koistinen,R., Oehninger,S., Patankar,M.S., Seppala,M. and Clark,G.F. (1996) J. Biol. Chem., 271, 3215932167.
Morrow,D.M., Xiong,N., Getty,R.R., Ratajczak,M.Z., Morgan,D., Seppala,M., Riitinen,L., Gewirtz,A.M. and Tykocinski,M.L. (1994) Am. J. Pathol., 145, 14851495.[Abstract]
Neurath,A.R., Jiang,S., Strick,N., Lin,K., Li,Y.Y. and Debnath,A.K. (1996) Nature Med., 2, 230234.[ISI][Medline]
Oehninger,S., Coddington,C.C., Hodgen,G.D. and Seppala,M. (1995) Fertil. Steril., 63, 377383.[ISI][Medline]
Pockley,A.G., Mowles,E.A., Stoker,R.J., Westwood,O.M.R., Chapman,M.G. and Bolton,A.E. (1988) J. Reprod Immunol., 13, 3140.[ISI][Medline]
Qin,B.Y., Bewley,M.C.,Creamer,L.K., Baker,H.M., Baker,E.N. and Jameson,G.B. (1998) Biochemistry, 37, 1401414023.[ISI][Medline]
Reddy,K.M.M.M., Karande A.A. and Adiga,P.R. (1992) Mol. Immunol., 4, 511516.
Romanos,M.A., Scorer,C.A. and Clare,J.J. (1992) Yeast, 8, 423488.[ISI][Medline]
Sarkar,G. and Sommer,S.S. (1990) Biotechniques, 8, 404407.[ISI][Medline]
Scorer,C.A., Buckholz,R.G., Clare,J.J. and Romanos,M.A. (1993) Gene, 136, 111119.[ISI][Medline]
Seppala,M., Jiang,S., Strick,N., Lin,K., Li,Y.Y., Koistinen,H., Koistinen,R. and Neurath,A.R. (1997) Lab. Invest., 77, 127130.[ISI][Medline]
Shida,K., Takamizawa,K., Nagaoka,M., Kushiro,A., Osawa,T. and Tsuji,T. (1994) J. Dairy Sci., 77, 930939.
Spector,A.A. and Fletcher,J.,E. (1970) Lipids, 5, 403411.[ISI][Medline]
Trimble,R.B., Atkinson,P.H., Tschopp,J.F., Townsend,R.R. and Maley,F. (1991) J. Biol. Chem., 266, 2280722817.
Waniska,R.D. and Kinsella,J.E. (1984) Int. J. Pept. Protein Res., 23, 573580.[ISI][Medline]
Received February 24, 2000; revised June 30, 2000; accepted December 26, 2000.