Production and Isolation of the Recombinant N-Lobe of Human Serum Transferrin from the Methylotrophic YeastPichia pastoris

PROTEIN EXPRESSION AND PURIFICATION ARTICLE NO.

8, 119–125 (1996)

0081

Production and Isolation of the Recombinant N-Lobe of Human Serum Transferrin from the Methylotrophic Yeast Pichia pastoris Anne B. Mason,*,1 Robert C. Woodworth,* Ronald W. A. Oliver,† Brian N. Green,†,‡ Lung-Nan Lin,§ John F. Brandts,Ø Beatrice M. Tam,\ Alexisann Maxwell,\ and Ross T. A. MacGillivray\ *Department of Biochemistry, University of Vermont, College of Medicine, Burlington, Vermont 05405; †Biological Materials Analysis Research Unit, Department of Biological Sciences, University of Salford, Salford M5 4WT, United Kingdom; ‡VG Organic Ltd., Altrincham, Cheshire WA 14-5R2, United Kingdom; §Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003; ØMicrocal, Inc., 22 Industrial Drive East, Northampton, Massachusetts 01060; and \Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

Received January 30, 1996, and in revised form March 26, 1996

The N-lobe of human serum transferrin has been expressed in the methylotrophic yeast Pichia pastoris by placing the hTF/2N cDNA under the control of the methanol-inducible alcohol oxidase promoter. Following induction with methanol, the N-lobe was efficiently secreted into a basal salt medium in shake flasks at a level of 150–240 mg/liter. As judged by mobility on SDS–PAGE, immunoreactivity with two domain-specific monoclonal antibodies, and both thermal stability and spectral properties (indictative of correct folding and ability to bind iron), the recombinant N-lobe produced by the yeast cells appears to be identical to that produced in a mammalian expression system. Electrospray-mass spectrometry and a third domain specific antibody, however, show that approximately 80% of the protein from the yeast cells contains one or two hexose residues. q 1996 Academic Press, Inc.

The transferrins are a family of glycosylated metalbinding proteins which function in the transport of iron to cells and as bacteriostatic agents in a variety of biological fluids (1–5). It is apparent from an examination of the amino acid sequence that a gene duplication event occurred during the evolution of the transferrins, resulting in the present day 80-kDa protein which binds two metal ions, tightly but reversibly, in homologous lobes. X-ray crystallographic studies reveal the 1 To whom correspondence should be addressed. Fax: (802) 8628229.

presence of two globular lobes each made up of two domains which define a deep cleft containing the binding site for each metal ion and the obligate synergistic anion, carbonate (6–14). In all transferrins for which the crystal structure has been determined, the ferric ion is directly coordinated to two tyrosine residues and to a single histidine and aspartic acid residue, as well as two oxygen atoms from the carbonate anion. The mature protein folds to bring these amino acids into proximity with each other. Expression of human serum transferrin by recombinant techniques presents a particular challenge due to the 19 disulfide bonds which must be properly joined to yield functional protein. On the other hand, the ability of recombinant transferrin to bind iron reversibly provides a relatively straightforward way of assessing function. Expression of human serum transferrin by bacterial systems has been very poor (15–18), (see Discussion). Recently expression of human serum transferrin in a baculovirus system was reported (19); maximum expression was 20 mg/ml. The most successful and versatile expression system to date has been a mammalian expression system developed and optimized in our laboratory (20–24). Nevertheless we undertook expression in yeast to reduce production cost and time and to pursue the production of totally 13C/15N-labeled protein for NMR studies. Here we report the successful expression and secretion of the N-lobe of human serum transferrin in Pichia pastoris and compare the properties of the yeast cell derived protein with those of the N-lobe produced in our baby hamster kidney (BHK) cell system. The results are compared to a report by Steinlein 119

1046-5928/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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et al. (25) which also describes the production of the Nlobe of transferrin in P. pastoris. MATERIALS AND METHODS

Materials. The chromatographic resin, Poros 50 HQ, and the Poros QE/M columns were from PerSeptive Biosystems. Centricon 10 microconcentrators and PM-10 ultrafiltration membranes were from Amicon. Rabbit anti-mouse immunoglobulin G was purchased from Southern Biological Associates. Na125I was from DuPont NEN. Baffled flasks and Dynatech removawells were obtained from a local distributor. All chemicals and reagents were analytical grade. In the present study, three monoclonal antibodies, which were specific to nonoverlapping epitopes in the N-lobe, were used; the antibodies have been thoroughly described previously (26). Two of the monoclonal antibodies, aHT / N1 and aHT / N2 were prepared in our laboratory while the third monoclonal antibody, HTF.14, was purchased from the Institute of Molecular Genetics at the Czechoslovakian Academy of Sciences. Yeast strain and culture media. The P. pastoris host strain used was GS115 (his4) obtained from Invitrogen Corporation as part of an expression kit which also included the pPIC9 secretion vector. All procedures and media formulae for yeast maintenance and growth were from the manual which comes with the kit except as noted below. The basal salt medium contained (per liter) 26.7 ml of 85% H3PO4 , 0.93 g CaSO4r2H2O, 18.2 g K2SO4 , 14.9 g Mg SO4r7 H2O, and 4.13 g KOH and was adjusted to pH 5.0 with NH4OH (27). In one production run, the acid was reduced to 5.0 ml/liter. The two media are referred to as high and low acid, respectively. Vector construction. The cDNA encoding for amino acids 1–337 of the mature human serum transferrin was cloned into the pPIC9 vector. The starting plasmid (BS HTF/N2 Xba/HindIII) contained part of the transferrin cDNA on a XbaI–HindIII fragment cloned into Bluescript, the cDNA coding for the transferrin signal sequence, residues 1–337, and two stop codons. This fragment had been used during the initial expression of hTF/2N in the pNUT vector (20). By performing a polymerase chain reaction (PCR) using BS HTF/N2 Xba/HindIII as template and the primers 5*HTFXhoI: 5* ATC CTC GAG AAA AGA GTC CCT GAT AAA ACT GTG AGA 3* 3*HTF: 5* CAC CAC AGC AAC AGC ATA ATA 3*, the signal sequence was removed and a cleavage site for KEX 2 was introduced just upstream of the codon for the amino-terminal valine residue of serum trans-

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ferrin. The KEX2 site is necessary for correct processing of the alpha factor-hTF/2N fusion protein during secretion in Pichia. The 5* oligonucleotide also introduces an XhoI site for subsequent cloning into the plasmid pPIC9. The 3* oligonucleotide is complementary to bp 370–390. After PCR, the fragment was cleaved with XhoI and BamHI (which cuts at nucleotide 354 of the cDNA) and the resulting fragment was cloned into the XhoI and BamHI sites of Bluescript to give the plasmid BS HTF/N2 XhoI/BamHI. The complete nucleotide sequence of the XhoI/BamHI fragment was determined to confirm the sequence at the 5* end of the cDNA and to ensure the absence of PCR-induced mutations. The plasmid was then cleaved with XhoI and NdeI (which cleaves at position 161 of the cDNA), and the XhoI/NdeI fragment was joined to the rest of the transferrin cDNA by replacement of the XhoI/NdeI fragment in BS HTF/2N Xba/HindIII giving the plasmid BS HTF/2N XhoI/HindIII. The N-lobe cDNA was then excised from this plasmid using XhoI and HincII and ligated into the XhoI and SnaBI sites of pPIC9. Colony selection. The construct designated pPIC9 HTF/N2 was digested with BglII and used to electroporate the GS115 strain of P. pastoris. The transformed cells were plated on medium deficient in histidine, thereby selecting only those yeast cells in which the pPIC9 vector had integrated into the chromosome. Colonies which grew only on the His0 plates were then screened for protein expression. Those which produced protein were further screened for their ability to utilize methanol by growing them on plates which contained methanol as the sole carbon source. Colonies which grow on these plates are designated mut/, while those that grow slowly or not at all are designated mut s or mut0. Expression of human N-lobe in P. pastoris shake flask culture. To start the growth phase, a single colony from an MD plate was taken to innoculate 10 ml of BMGY in a 50-ml conical tube. Following incubation overnight at 307C with vigorous shaking, the culture was used to seed two 2-liter baffled flasks containing 500 ml each of BMGY. After overnight incubation as before, 25 ml of 101 glycerol was added to each flask and the incubation was continued for an additional 24 h. To begin the induction phase, the yeast cells were harvested by centrifugation and resuspended in 500 ml of basal salt solution containing 0.5 ml of PTM-1 salts (28) and 2.5 ml of methanol in a 1-liter baffled flask. Each day, 2.0 ml of methanol was added to the flask and an aliquot of the culture medium was taken for analysis. On the third or fourth day the yeast cells were harvested and resuspended as above in 500 ml of fresh solution. The supernatant (17 induction) containing the recombinant protein was collected and frozen. Following another 4–5 days of incubation as above, the super-

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RECOMBINANT TRANSFERRIN HALF-MOLECULE PRODUCTION

natant (27 induction) was collected and the yeast cells were discarded. The supernatants from both inductions were combined and the pH was adjusted to 7.0 with 10 N NaOH. The white precipitate which resulted was removed by filtration through No. 1 Whatman filter paper. The yellow supernatant was reduced in volume to less than 50 ml using an Amicon stirred cell using a PM-10 membrane and exchanged into 10 mM Tris– HCl, pH 8.0, containing 0.02% sodium azide. Isolation of recombinant human N-lobe. Slightly more than one equivalent of ferric nitrilotriacetate (molar ratio 1:2) was added to saturate the transferrin in the medium as determined by the radioimmunoassay. Following centrifugation to clarify the sample, the protein was applied to a Poros 50 HQ column (2.5 1 12 cm) using a Pharmacia P-1 pump at a rate of approximately 10 ml/min. The recombinant N-lobe was eluted from the column with a single step consisting of 180 mM Tris – HCl, pH 8.0, containing 0.02% NaN3 . Final purification was on a Poros QE/M (10/100) column run on a PerSeptive Biosystems Sprint chromatography system. The column was equilibrated and run in 50 mM Tris/bis – Tris propane, pH 8.0, at a rate of 7 ml/min. A linear gradient of 0 – 400 mM NaCl in the same buffer over 5 column volumes was used to develop the column. The Sprint system allows simultaneous monitoring of the pH, the conductivity, and the absorbance at 280 nm. Fractions of 3 ml were collected. SDS–PAGE (12% separating gel with 4% stacker) was performed to monitor protein production and purity using a mini-Protean II slab cell apparatus from BioRad. The gels were cast and run according to the manufacturer’s instructions. A competitive solid-phase immunoassay (21) was used to determine the concentration of recombinant Nlobe in the yeast cell medium and at various stages of the purification. Visible absorption spectra were recorded on a Cary 219 spectrophotometer as described (22). The N-terminal sequence of the first six amino acids was determined by Dr. Steve Smith of the Protein Chemistry Laboratory at the UTMB Cancer Center (Galveston, TX). Details of the mass spectrometry analyses have been given (22,23). Similarly, a complete description of the differential calorimetry scans is found in our previous work (29). RESULTS

As described under Materials and Methods, manipulation of the cDNA for human serum transferrin led to a plasmid which contained the coding region for the Nlobe comprising residues 1–337 under the control of the AOX1 promoter. The hTF/2N cDNA was cloned into

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FIG. 1. Production rate for primary (A) and secondary (B) inductions of two different batches of recombinant hTF/2N secreted into basal salt media by the yeast P. pastoris. Batch 1 (j) had high acid and batch 2 (l), low acid. Each sample was assayed twice. The error bars are the standard of the mean for the two assys.

Bluescript at unique XbaI (5*) and HindIII (3*) sites. A 5* XhoI site was created using PCR to allow cloning into the XhoI site of pPIC9. In addition, following the XhoI site the coding sequences for Lys and Arg residues were introduced to recreate the Saccharomyces cerevisiae a-mating factor pre–pro leader sequence. The native signal sequence was deleted from the construct. Initial production runs were performed in BMMY medium, but purification of the secreted recombinant protein from this medium was found to be difficult and the recoveries were low. Protein production in the basal salt medium was found to be equivalent to that in BMMY medium and the downstream purification was simpler, with better recoveries of the hTF/2N. Typical production runs (one in high acid and one in low acid) are shown in Fig. 1. A recurrent feature of the yeast system has been difficulty in getting reproducible results from the radioimmunoassay as reflected in the standard deviations on the two curves. There was more variability than found in the assays routinely done to monitor production of the N-lobe expressed by baby hamster kidney cells (21). Nevertheless the general

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Recovery of Recombinant hTF/2N from the Supernatant of Yeast Cells Containing the Expression Vector a

Step

FIG. 2. Production of recombinant hTF/2N as monitored by SDS– PAGE under reducing conditions. Samples (5 ml of culture supernatant) were electrophoresed from the primary induction, Days 1–4 (lanes 1–4) and from the secondary induction, Days 1–5 (lanes 6– 10). Bio-Rad low-molecular-weight standard was run in lane 5. From top to bottom, molecular weights, are 97.4, 66.2, 45, 31, 21.5, and 14.4 kDa. The samples and standards were visualized with Coomassie blue.

trends were very reproducible and show almost linear production of recombinant N-lobe over the period of both 17 and 27 production. Linear regression analysis generally gives correlation coefficients greater than 0.97 for the hTF/2N concentration (mg/ml) plotted against days in culture. Increasing protein production is further substantiated by the results shown in Fig. 2, in which 5 ml of medium from each day was electrophoresed on a SDS gel. Samples were from run 1 shown in Fig. 1. In five separate production runs 52.2 ({5.8) g of yeast (wet weight) yielded 177.0 ({25.2) mg/ml of N-lobe in the primary induction of 4 days and 203.8 ({25.8) mg/ml of N-lobe in the secondary induction of 3–5 days. The maximum production reached was approximately 230 mg/ml and was obtained in both the high and low acid basal media. The gel shows that the recombinant hTF/2N is virtually the only protein in the yeast cell medium. Nevertheless, chromatography was required to eliminate other nonproteinaceous matter which at first masks the pink color of the iron loaded N-lobe. The medium is yellowishgreen in color and has a substantial absorbance at 280 nm. Although the Poros 50 column yields material which appears to be pure, chromatography on the QE/M column was included in the protocol to assure that the final purified sample was comparable to the N-lobe produced in our mammalian expression system (see Discussion). The recovery of the recombinant hTF/2N from the yeast medium improved with each successive collection, leveling off at approximately 70%. The recovery of N-lobe during purification of the sample collected from batch 2 in Fig. 1 is given in Table 1. Protein characterization. The hTF/2N isolated from the QE/M column was analyzed and compared to the N-lobe produced by baby hamster kidney cells using

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Volume (ml)

Total A280

hTF/2N (mg)

Yield (%)b

775

1624

179

100 (R)

900

1125

139

78 (R)

55 40 70

380 172 154

NDc 136 123

ND 75 (S) 69 (S)

Original culture supernatant Neutralization/ filtration PostAmicon/ PrePoros PostPoros50 Post QE/M

a Details of the purification procedure are given under Materials and Methods. b R refers to the amount of hTF/2N determined by radioimmunoassay. S refers to the amount of recombinant hTF/2N determined by spectral analysis. c Not determined.

the pNUT vector (20,21). First, the elution profiles on the QE/M column were identical (data not shown). Second, the samples appear identical on SDS–PAGE (Fig. 3). In addition, both proteins bind iron as indicated by their salmon pink color which gives an absorbance maximum in the visible spectrum at 465 nm. Characteristic spectra from the BHK and Pichia derived hTF/ 2N were indistinguishable. Furthermore, the iron could be removed and each protein could be retitrated with iron to the previous level. Reversible iron binding is a benchmark of function for transferrin and strongly suggests that both of the recombinant proteins are correctly folded. Identical extinction coefficients were obtained from titration of each protein with cobalt (30).

FIG. 3. Comparison of electrophoretic behavior of recombinant hTF/2N produced by the yeast P. pastoris (lanes 1 and 4) and by baby hamster kidney cells (lanes 2 and 5). The samples in lanes 1 and 2 are reduced; those in lanes 4 and 5 are not reduced. A BioRad molecular weight standard was run in lane 3. Molecular masses are given in the legend to Fig. 2. Approximately 3 mg of each sample was loaded onto the gel. Visualization was by staining with Coomassie blue.

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RECOMBINANT TRANSFERRIN HALF-MOLECULE PRODUCTION TABLE 2

Electrospray Mass Analysis of Two Batches of Human Serum Transferrin N-lobe Produced in the Yeast P. pastoris

hTF/2N

Calculated Mr

Experimental Mr

Relative abundance (%)

Batch 1

37,151

Batch 2

37,151

37,152 37,314 37,475 37,151 37,314 37,475

20 100 10 20 100 4

D Mr /1 /163 /324 0 /163 /324

nominal 10 mg/ml hTF/2N from the yeast cells with the BHK-produced hTF/2N as the standard gave different results for the three antibodies and appears to allow an accurate estimate of the amount of recombinant protein which contains carbohydrate. If one assumes that the presence of one or two hexoses on the protein blocks the binding of the antibody, aHT / N1 , then 18% of the protein is carbohydrate free. This also assumes that there is a single attachment site. This assumption seems warranted in view of the close agreement of results from the mass analysis and the radioimmunoassay. DISCUSSION

Further support comes from dsc studies in which the apparent Tms (the temperature at which one half of the total area has been generated) were identical for the hTF/2N produced by the yeast cells and by the BHK cells, both 867C. The apparent Tm for the apo-proteins was 677C for the yeast derived N-lobe and 66.47C for the BHK protein. Amino terminal sequence analysis established that the first six amino acids are Val-Pro-Asp-Lys-Thr-Val. This is the N-terminal sequence reported for holo-hTF (31,32) and for hTF/2N produced in BHK cells (20). The result is important in confirming the correct processing of the alpha factor-hTF/2N fusion protein inside the yeast cells. Results from electrospray mass analysis of two different preparations of N-lobe from P. pastoris are presented in Table 2. As the N-lobe of human serum transferrin is not glycosylated the calculated Mr for the Nlobe composed of residues 1–337 is 37,151. However, a large portion of the recombinant protein is larger by 163 amu. A small amount is larger by twice this mass (324 amu), while an intermediate amount is the correct molecular mass. These higher mass forms are consistent with the yeast cells adding hexose and dihexose residues to the majority of the recombinant protein. It should be noted that in the absence of measurements of the relative ion abundances (RA%) of mixtures of known composition, the composition of the prepartions inferred from the measured RA given in Table 2 may not be strictly correct. However, from previous analytical studies of other glycoproteins we believe that they are the correct order of magnitude. While monitoring the production of the recombinant N-lobe from the yeast cells using our solid-phase radioimmunoassay, a discrepancy was found between the amount of protein observed by electrophoresis and the results of the assay with the monoclonal antibody aHT / N1 . There appeared to be more protein on the gel than predicted by the assay. As shown in Table 3, this observation has been confirmed. Measurement of a

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Functional human serum transferrin N-lobe has been successfully produced from the methylotrophic yeast P. pastoris grown in shake flasks, at levels exceeding 200 mg/ml. The production level is almost twice that realized in our mammalian cell expression system where a maximum of 120 mg/ml has been obtained. Advantages of the yeast cell system include that the purification of the recombinant N-lobe is simpler and the recovery of purified protein is equivalent. The cost is considerably less and there is a potential advantage in the complete elimination of serum and serum substitutes. Possible disadvantages include the presence of one or two carbohydrate residues on most of the N-lobe produced in the yeast system. Also, since the yeast cells make all their own amino acids, labeling of the recombinant protein with specifically labeled amino acids is unlikely to be successful. In contrast, specifically labeled 13C, 15N, and 3H amino acids have been incorporated into the N-lobe produced in our BHK cell system allowing NMR experiments to investigate the role of specific residues in protein function (33). The present report confirms and extends the findings of Steinlein et al. (25), i.e., that functional N-lobe of human serum transferrin can be produced by the yeast P. pastoris. It differs in some technical details which lead to yields in our work which are approximately

TABLE 3

Determination of the Concentration of Human Transferrin N-lobe from the Yeast P. pastoris by Solid Phase Radioimmunoassay with Three Monoclonal Antibodies Which Recognize Different Epitopes in the N-lobe of hTF

Monoclonal antibodya

Concentration of a nominal 10 mg/ml solution

HTF.14 aHT / N1 aHT / N2

11.2 { 0.5 1.9 { 0.4 10.2 { 0.3

Note. Human hTF/2N from BHK cells was used as a standard. a The antibodies are designated as described (26).

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fivefold higher in five different production runs. Our purification scheme is easier, faster, and allows 70% recovery of recombinant N-lobe vs 28% reported by Steinlein et al. The more extensive characterization showing that there is carbohydrate on much of the protein produced by the yeast is an important finding which may make the yeast system unsuitable for certain applications. Among the reasons for undertaking expression of the human transferrin N-lobe in the yeast system were cost, simplicity of downstream purification of the recombinant protein, and the desire to make totally 13C/ 15 N-labeled protein for nuclear magnetic resonance studies. We also regard production of the N-lobe by yeast cells as a first step to future production of fulllength hTF and eventually of the C-lobe of hTF which has proved extremely difficult to produce in any of the expression systems which have been tried. Because we wished to develop a system to allow 13C/ 15 N labeling, as described by Laroche et al. for tick anticoagulant protein (TAP) (34), we wanted to use the simpler shake flask system rather than using a fermentor. Clearly, not all the variables have been explored in great detail. The present protocol works in a predictable and reproducible manner and will yield enough N-lobe for the projected NMR studies. An important finding in this regard is that yields from the low acid medium were equal to those in high acid medium used in the earlier studies. Since the pH is adjusted to 5.0 with NH4OH, which is the sole source of nitrogen, the low acid medium obviously requires far less 15NH4OH to reach the desired pH. Using an expensive 13C/15N-labeled algal extract, we were able to produce only about 5 mg of hTF/2N in the BHK system before one or more of the amino acids became ratelimiting (unpublished observations). The yeast system appears to be much more promising for this type of labeling. An interesting feature of the yeast cell expression, alluded to above is the low pH of the yeast medium, which appears to be a potentially hostile environment for the newly secreted recombinant protein. The low pH almost certainly precludes iron binding, which greatly stabilizes hTF. Nevertheless, the almost linear increase in the amount of recombinant hTF/2N seen in Fig. 1 implies that the protein produced is not degraded. As noted below, the recently described bacterial expression system suffered from loss of recombinant protein due to extensive proteolytic breakdown (18). One of the factors contributing to the variability in results from the assay is almost certainly the pH and the fact that the recombinant N-lobe may be all or partially iron free. The three domain-specific monoclonal antibodies used in this study show a slight preference for the iron form of hTF (26). The maximum secretion reported for a heterologous

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protein in P. pastoris is 1.7 gm/liter for the small (60 amino acid) tick anti-coagulant protein (34). This level was achieved in batch-fed fermentation where the culture conditions are carefully monitored and controlled. In ‘‘high culture density’’ shake flasks, the maximum production of TAP was 72 mg/liter. Thus the levels obtained in our shake flasks appear to be quite respectable. Although there have been at least three reports of the production of human serum transferrin and/or the N- and C-lobes of transferrin in bacterial systems, the limited amount of functional protein which is obtained appears to make this approach impractical (15–18). In the most recent study, a maximum of 60 mg/liter of hTF is reported (18). The protein has Met at the Nterminus (instead of the Val found in the native hTF) and although a ‘‘brown-pink’’ hue is observed in the cells there is no direct evidence that the recombinant protein can bind iron. In fact it is reported that 95% of the protein produced is in the insoluble fraction. Problems with both proteolysis and inhibition of bacterial growth were encountered. The yeast system described in the present report is capable of producing fully functional protein at levels which are three- to fourfold higher than realized in the bacterial system. No refolding protocols are required. Clearly the yeast cell system is superior in the production of usable product. We are currently extending these studies to include the expression of full-length hTF and the C-lobe in P. pastoris. ACKNOWLEDGMENTS This work was supported by USPHS Grant R01 DK 21739 from the National Institute of Diabetes and Digestive and Kidney Diseases.

REFERENCES 1. Brock, J. H. (1985) Transferrins, in ‘‘Metalloproteins: Metal Proteins with Non-redox Roles’’ (Harrison, P., Ed.), pp. 183–262, MacMillan, London. 2. Harris, D. C., and Aisen, P. (1989) Physical biochemistry of the transferrins, in ‘‘Iron Carriers and Iron Proteins’’ (Loehr, T. M., Ed.), pp. 239–351, VCH Publishers, New York. 3. Aisen, P. (1989) Physical biochemistry of the transferrins: Update, 1984–1988, in ‘‘Iron Carriers and Iron Proteins’’ (Loehr, T. M., Ed.), pp. 353–371, VCH Publishers, New York. 4. Thorstensen, K., and Romslo, I. (1990) The role of transferrin in the mechanism of cellular iron uptake. Biochem. J. 271, 1–10. 5. Chasteen, N. D., and Woodworth, R. C. (1990) Transferrin and lactoferrin, in ‘‘Iron Transport and Storage,’’ (Ponka, P., Schulman, H. M., and Woodworth, R. C., Eds.) pp. 68–79, CRC Press, Boca Raton, FL. 6. Anderson, B. F., Baker, H. M., Dodson, E. J., Norris, G. E., Rumball, S. V., Waters, J. M., and Baker, E. N. (1987) Structure of ˚ resolution. Proc. Natl. Acad. Sci. human lactoferrin at 3.2 A USA, 84, 1769–1773. 7. Anderson, B. F., Baker, H. M., Norris, G. E., Rice, D. W., and Baker, E. N. (1989) Structure of human lactoferrin: Crystallo˚ resolution. graphic structure analysis and refinement at 2.8 A J. Mol. Biol. 209, 711–734.

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pepa

AP: PEP