High-Level Expression and Purification of Coffee Bean α-Galactosidase Produced in the YeastPichia pastoris

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 324, No. 1, December 1, pp. 65–70, 1995

High-Level Expression and Purification of Coffee Beana-Galactosidase Produced in the Yeast Pichia pastoris1

Alex Zhu, Catherine Monahan, Zhanfan Zhang, Rosa Hurst, Lin Leng, and Jack Goldstein2

Lindsley F. Kimball Research Institute of The New York Blood Center, 310 East 67 Street, New York, New York 10021

Received July 14, 1995, and in revised form August 15, 1995

from glycoconjugates (1). Coffee bean a-galactosidase,a-Galactosidase isolated from coffee beans cleaves which functions as a monomer of approximately 41

the terminal a-galactose residues from oligosaccha- kDa, apparently exists as two forms (I and II) withride chains on blood group B red cells, thus generating different isoelectric points and pH optima (2). However,group O cells. Such enzymatically converted red cells the molecular basis for the differences observed withnot only maintain full erythrocyte integrity and viabil- these two forms has yet to be elucidated. The enzymeity in vitro, but also demonstrate immune tolerance demonstrates a relatively broad substrate specificity,and a normal life span in vivo. In order to produce cleaving a variety of terminal a-galactosyl linkages in-large quantities of recombinant a-galactosidase for cluding blood group B antigens on the red cell surfaceuse in the study of blood-type conversion, we sub- (3, 4). The B antigen is composed of a terminal galac-cloned the cDNA coding for coffee bean a-galactosi- tose residue a-linked to an assembled blood group Odase into the EcoRI site of the vector pPIC9 in order carbohydrate chain on membrane glycoproteins andto express the enzyme in Pichia pastoris, a methylo- glycolipids. Treatment of type B red cells with coffeetrophic yeast strain. After P. pastoris transformation, bean a-galactosidase results in a specific removal ofcolonies were screened for high-level expression of a-

the terminal a-galactose residues, thus generating se-galactosidase, based on enzyme activity. In order torologically type O red cells (5). Such enzymatically con-increase enzyme production, the growth conditions inverted red cells not only maintain full erythrocyte in-the shake flask culture and fermentor culture weretegrity and viability in vivo, but also demonstrate im-optimized. Under the conditions applied, biologicallymune tolerance and a normal life span in vivo (6).active a-galactosidase was produced and secreted into

We have previously reported cloning the cDNA cod-the culture medium at a level of approximately 0.4 ging for coffee bean a-galactosidase and expressing bio-per liter of the fermentor culture. The protein was pu-logically active enzyme in insect cells with a baculovi-rified to apparent homogeneity by a simple chromatog-

raphy procedure, as suggested by a single band of 41 rus vector (7). However, the production of recombinantkDa on sodium dodecyl sulfate–polyacrylamide gel a-galactosidase in the baculovirus expression systemelectrophoresis. Its homogeneity was further con- is time consuming and expensive, which limits its ap-firmed by chromatofocusing and N-terminal sequenc- plication for large-scale production of the enzyme useding. P. pastoris appears to be the choice as host for the in the B r O blood-type conversion procedure. In anlarge-scale production of recombinant a-galactosidase attempt to establish a more efficient system for produc-used for blood type conversion. q 1995 Academic Press, Inc. tion of coffee bean a-galactosidase, we subcloned the

cDNA into a vector for expression in Pichia pastoris,a methylotrophic yeast strain, that has been recentlydeveloped as a host for high-level heterologous proteina-Galactosidase (EC 3.2.1.22) is an exoglycosidaseexpression (8). The level of recombinant enzymewidely distributed in nature and is responsible for inreached as high as 400 mg per liter of the culture me-vivo cleavage of a-linked terminal galactose residuesdium, suggesting that P. pastoris is the host of choicefor overexpressing recombinant a-galactosidase. In

1 Supported in part by Office of Naval Research Grant N00014- contrast human a-galactosidase was expressed in in-91-J-1180 with funds provided by the Naval Medical Research and sect Sf9 cells (9) and in CHO cells (10) at a level of 5.6Development Command to the New York Blood Center.

mg/liter and 0.175 mg/liter/day, respectively, and a-2 To whom correspondence should be addressed. Fax: 212-879-0243. galactosidase cDNA, isolated from the plant Cyamposis

650003-9861/95 $12.00Copyright q 1995 by Academic Press, Inc.All rights of reproduction in any form reserved.

/ m4496$9195 10-26-95 10:29:00 arca AP: Archives

66 ZHU ET AL.

FIG. 1. The 5* cloning site of the plasmid paF-BZ. EcoRI (underlined sequence) of the vector was used for the cloning of a-galactosidasecDNA. The shaded sequence is part of the signal peptide of yeast a mating factor and the boxed peptide indicates the beginning of themature a-galactosidase. Arrows indicate the signal cleavage sites (see text).

brane was then transferred onto the surface of a MM plate for thetetragonoloba was expressed in Hansenula polymorphaenzyme induction. After incubation at 307C for 16 h, the nitrocellu-at the level of 42 mg/liter (11).lose membrane was placed on a Whatman No. 4 filter saturatedIn this paper we describe our development of a high- with the chromogenic a-galactosidase substrate 5-bromo-4-chloro-3-

level expression system for recombinant coffee bean indolyl-a-D-galactopyranoside (1 mg/ml in phosphate buffer, pH 6.5).Those colonies positive for a-galactosidase activity were visualizeda-galactosidase in P. pastoris and purification of theby a change in color from yellow to blue, usually within 15 min afterenzyme by a simple chromatography procedure.exposure to the substrate at room temperature.

Second phase screening. The colonies identified as producing ac-METHODStive a-galactosidase were individually inoculated in 5 ml BMGY andgrown at 307C, 300 rpm, for 2 days in 50-ml conical tubes (FalconCell Strain and Culture Media2098). The yeast cells were harvested by centrifugation at 1500g for

The P. pastoris host strain, GS115 (his40), was purchased from 5 min at room temperature and resuspended in BMMY at half ofInvitrogen. The media used for the P. pastoris in shake flask culture the original culture volume. After incubation for 1 day (16–18 h) atare outlined by Invitrogen and include: minimal dextrose medium 307C and 300 rpm, both cell density and a-galactosidase activity were(MD),3 minimal methanol medium (MM), buffered minimal glycerol- quantitated.complex medium (BMGY), buffered minimal methanol-complex me-dium (BMMY), and regeneration dextrose base (RDB). The fermentorculture medium contains 2.3% H3PO4, 5 mM CaSO4, 60 mM MgSO4, Culture Conditions for Shake Flask Culture70 mM KOH, 100 mM K2SO4, and 4% glycerol (pH 4.5). Trace saltsolution contains 8 mM CuSO4r5H2O, 0.5 mM NaI, 20 mM MnSO4, The P. pastoris clone, paF-BZ-22, which produced the highest level0.8 mM Na2MoO4r2H2O, 0.3 mM H3BO3, 2 mM CoCl2, 51 mM ZnCl2, of a-galactosidase as determined by the second phase screening pro-80 mM FeSO4r7H2O, 0.8 mM biotin, 1 ml H2SO4 (personal communi- cedure, was used for all shake flask and fermentation studies. Thecation of R. A. Brierley at Cephlon, Inc.). shake flask cultures were grown in baffled flasks with BMGY me-

dium by incubation in a rotary shaker at 300 rpm and 307C.

Methanol induction. After a culture of paF-BZ-22 was grown forPlasmid Construction and P. pastoris Transformation3 days, methanol was added to a final concentration of either 1 or

cDNA coding for mature a-galactosidase (7) was subcloned into 5%. The same amount of methanol was added each day for up to 4the EcoRI site of P. pastoris expression vector pPIC9, generating the days. The cell density and a-galactosidase activity were monitoredplasmid paF-BZ. The plasmid sequence around the 5* cloning site of daily.the insert is shown in Fig. 1. The recombinant enzyme is expressed

pH variation. After paF-BZ-22 was grown in BMGY (pH 7.0) foras a fusion protein and secreted into the culture medium via the3 days, the medium was adjusted to pH 5.5, 6.5, or 7.5 with NH4OHsecretion signal derived from yeast a factor. The plasmid paF-BZor H3PO4, prior to the addition of methanol to a final concentrationwas digested with either SalI, for integration at the HIS4 locus (His/of 5%. Samples for cell density and a-galactosidase activity determi-Mut/), or BglII, for integration at the AOX1 locus (His/ Muts) (Mut/nation were extracted daily.and Muts are wild type for methanol utilization and methanol utiliza-

tion slow, respectively). P. pastoris transformation with digestedplasmid was carried out using the spheroplast method as outlined

Culture Conditions for Fermentor Cultureby Invitrogen.

The inoculum for each fermentation run was generated by growingFunctional Selection of Transformants Expressing a 300-ml YNB culture in 2-liter baffled shake flasks at 300 rpm, 307C

until cell density reached 5 1 108 cells/ml or greater (approx. 2–3a-Galactosidasedays). Each fermentation run was initiated by adding 200 ml of

First phase screening. Transformed colonies growing only on the inoculum shake flask culture to 4 liters of fermentation culture me-surface of the top agar of RDB plates were transferred onto nitrocel- dium supplemented with 4.35 ml trace salts solution per liter ofluose membrane (0.45-mm pore size, Schleicher & Schuell) by replica culture medium; thus, inoculum is 5% of total volume. In the firstplating. The membrane was placed on a MD plate and the colonies phase of fermentation, the cell density reached 170–200 g wet cellwere grown directly on the membrane, overnight at 307C. The mem- wt per liter after feeding for approximately 1 day with 50% glycerol

supplemented with 4.35 ml trace salts solution per liter of glycerolfeed. In the second phase, the cell density reached 230 g/liter afterfeeding 100% methanol supplemented with trace salts for 3 days at3 Abbreviations used: MD, minimal dextrose medium; MM, minimal

methanol medium; BMGY, buffered minimal glycerol-complex me- a rate of 3 ml/h/liter. The a-galactosidase activity reached 10-12U/ml at the conclusion of the fermentation run. Throughout, thedium; BMMY, buffered minimal methanol-complex medium; RDB, re-

generation dextrose base; YNB, yeast nitrogen base medium; SDS– temperature was kept constant at 307C, the agitation was 1000 rpm,and the condensed air flow was kept at 20%.PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.


67EXPRESSION OF COFFEE BEAN a-GALACTOSIDASE IN Pichia pastoris

Copy Number Determination mately 200 His/ Mut/ colonies were produced from P.pastoris transformation with 1.4 mg of SalI-digestedThe number of copies of the a-galactosidase gene incorporatedpaF-BZ and were screened for high enzyme activity byinto the genome was determined by DNA dot blot hybridization.

Genomic DNA was extracted from paF-BZ-22 and the parental the two-phase process described under Methods. Theyeast strain GS115 using the genome DNA isolation kit (Bio101). colonies were grown and induced with methanol di-The URA3 gene, which is present as a single copy in the P. pastoris rectly on the nitrocellulose membrane. Approximatelygenome, was used as an internal control. Two probes, BZ and URA,

half of the colonies expressed the recombinant enzymespecific for a-galactosidase and URA3 sequence, respectively, wereas suggested by a blue color during first phase screen-derived from the plasmid pYE-BZ (a-galactosidase cDNA sub-

cloned into the vector pYES-2, Invitrogen) and labeled by a 32P ing. Screening for a-galactosidase activity of coloniesrandom primer method (Amersham RPN). The genomic DNAs on the nitrocellulose membrane is a very efficient(1.5–5 mg) were denatured and then applied to the dot blot appara- method to identify positive colonies from the originaltus in duplicate according to the procedure of Clare et al. (12).

plate, while eliminating the negative colonies from fur-Each blot was hybridized with a different probe and relative radio-ther analysis. The a-galactosidase activity level variedactivity of these two probes was calibrated by hybridization to the

plasmid pYE-BZ. After autoradiography, each of the labeled dots greatly among colonies due to the different number ofwas counted in a scintillation counter to calculate the copy number copies of the a-galactosidase gene integrated into theof the a-galactosidase gene in paF-BZ-22. P. pastoris genome during transformation. In order to

quantitatively compare the level of a-galactosidase ex-Sample Preparation and Chromatography pression, the colonies which gave more intense blue

After enzyme induction in the fermentor was complete, cells were color in the first phase of screening were chosen forremoved by centrifugation at 3500g for 30 min. The a-galactosidase growth in shake flasks during second phase screening.containing supernatant was concentrated and equilibrated with 20 The colony, paF-BZ-22 (His/ Mut/), which expressedmM NaAc buffer, pH 4.1, using a Mr 10,000 cutoff ultrafiltration

the highest level of a-galactosidase activity (0.45 U/membrane (Millipore Corp.). After spinning at 13,000g for 30 min,ml after 1 day of induction) was chosen for furtherthe supernatant containing recombinant a-galactosidase was applied

to a cation-exchange column, Macro Prep S-50 (Bio-Rad Labora- experimentation, including all of the shake flask andtories) which had been equilibrated with 20 mM NaAc buffer. The fermentation studies.column (volume, 100 ml) was then washed with 1500 ml of the same The variation of a-galactosidase activity levels in dif-buffer followed by 1000 ml of the buffer plus 50 mM NaCl. Finally

ferent transformed colonies suggested that multiplea-galactosidase was eluted from the column with a linear NaCl gradi-copy integrations in the P. pastoris genome had takenent ranging from 50 to 350 mM.place. In order to determine the copy number of the a-galactosidase gene in paF-BZ-22, three independentEnzyme Assaydot blot hybridization procedures were carried out,

a-Galactosidase activity was measured by incubating the sample each using the same extracted genomic DNA from paF-with 1.25 mM p-nitrophenol-a-galactopyranoside at pH 6.5 and 267C.BZ-22 but different probe preparations. The parentalThe reaction was terminated by adding 0.1 M borate buffer, pH 9.8,strain GS115 was used as a negative control. The copyand measured at 405 nm. One unit (U) of activity is defined as the

amount of a-galactosidase that hydrolyzes 1 mmol of substrate per number in the paF-BZ-22 genome was estimated atminute under the assay conditions. 11 copies by averaging the data from three dot blot

hybridization experiments (copy numbers 7.7, 11.2,N-Terminal Amino Acid Sequence and 13.0).

Purified recombinant a-galactosidase was concentrated to 11.3 mg/ml and 40 mg of a-galactosidase was loaded to ABI 477A/120A se- Optimization of the Growth Conditions in Shakequencer for amino acid sequence analysis.

Flask and Fermentor

In order to optimize the growth conditions for shakeRESULTSflask culture, we varied the method of methanol induc-

Selection of P. pastoris Transformants with a High- tion and pH of the culture medium. The recombinantLevel Expression of a-Galactosidase protein was induced by varying the feeding method

(once every 24 h or one-time induction at Day 0) andThe transformation efficiency, using SalI-digestedpaF-BZ was compared to BglII-digested paF-BZ in P. the amount of methanol added (1 or 5% final concentra-

tion). As shown in Fig. 2A, the highest level of a-galac-pastoris spheroplast transformation. The data indi-cated that the average number of His/ Mut/ colonies tosidase activity was observed either by the one-time

addition of 5% methanol at Day 0 of induction or bygenerated by integrating at the HIS4 locus (SalI diges-tion) was 20-fold higher than the average number of feeding 1% methanol every 24 h; therefore, we chose

the 5% methanol induction in all other shake flaskHis/ Muts colonies generated with AOX1 locus integra-tion. Such a discrepancy in transformation due to dif- studies. The a-galactosidase activity did not increase

appreciably upon induction for periods longer than 4ferent locus integration is consistent with the observa-tions made by Scorer et al. (13). A total of approxi- days (data not shown). In addition, it appeared that the


68 ZHU ET AL.

0.5-unit intervals. The aliquots were then incubated at307C for up to 7 days. At the end of incubation, almost100% of the a-galactosidase activity remained with thealiquots at pHs from 4 to 5, whereas those aliquotsincubated at lower or higher pHs showed reduced en-zyme activity (Fig. 3). As a result we carried out cellgrowth and enzyme induction in the fermentor at pH4.5. Under the experimental conditions, after 4 daysinduction with methanol, the level of a-galactosidasesecreted into the culture medium reached approxi-mately 12 U/ml or 400 mg/ml. Further induction didnot lead to higher enzyme production.

Purification of a-Galactosidase ProducedFIG. 2. The a-galactosidase activity in shake flasks culture grownin P. pastorisand induced under different conditions. The P. pastoris culture (paF-

BZ-22) was grown in BMGY medium at 307C for 3 days. The culture After removal of the P. pastoris cells from the fermen-(É8 1 108 cells/ml) was equally divided and each was induced with

tation culture, the a-galactosidase containing superna-methanol under different conditions. (A) various methanol feedingtant was concentrated and subjected to chromatogra-schemes: (l) 1% methanol added at Day 0, (l) 1% methanol added

daily, (j) 5% methanol added at Day 0, (m) 5% methanol added phy with a strong cation exchanger (macro-prep S50).daily. (B) various pH values for the culture media: (j) pH 5.5, (l) The binding capacity of the column was more than 25pH 6.5, (m) pH 7.5. mg of a-galactosidase per milliliter of S-50 resin. After

the unbound proteins were washed off, a linear NaClgradient ranging from 50 to 350 mM was applied. Bothcell growth was not affected by the amount of methanol the absorbancy at 280 nm and the a-galactosidase ac-added. The initial cell density was 8 1 108 and in- tivity from each fraction of the gradient were measuredcreased to 2.5 1 109 over the entire induction period, as illustrated in Fig. 4. Under the experimental condi-regardless of the method of induction. tions, the a-galactosidase molecule in the supernatantAlthough P. pastoris cells grew normally at the pHs was bound to the column and efficiently eluted by atested, the level of a-galactosidase produced was af- NaCl gradient. The eluate was analyzed by SDS–fected by pH of the culture medium (Fig. 2B). At pH

7.5 the level of a-galactosidase was significantly lowerthan that from the cultures grown at pH 5.5 or pH6.5 during the entire induction period. Although thecultures at pH 5.5 and 6.5 demonstrated similar a-galactosidase activity until the third day of induction,the activity in the pH 5.5 culture dropped, while theactivity in the pH 6.5 culture continued to increase bythe end of the fourth day. Therefore, the culture me-dium at pH 6.5 seems to offer the most appropriateconditions for a-galactosidase production in shake flaskculture.

In order to produce recombinant a-galactosidase inlarge quantities, we carried out P. pastoris inductionin a fermentor. Conditions for high cell-density fermen-tation, such as the pH requirement, were different fromthose used for the shake flask cultures. Although cellscan grow in a wide range of pHs from 3 to 7 in a fer-mentor (8), the level of expressed protein is affected bythe pH of the culture medium. This is primarily due tothe stability of the expressed protein under a prolongedinoculation period and activity of host proteases at dif-

FIG. 3. Effect of pH on the stability of the recombinant a-galactosi-ferent pHs. Because of the nature of individual pro-dase in the fermentor culture supernatant. The induced culture fromteins, the best conditions for a-galactosidase produc-the fermentor was centrifuged to remove the cells. The supernatanttion in P. pastoris had to be determined experimentally. was equally divided and adjusted to pH values ranging from 3.13 to

The induced culture supernatant containing a-galac- 5.48. The enzyme activity (U/ml) was monitored during the 7-dayincubation period at 307C.tosidase was adjusted to pHs ranging from 3 to 7 at


69EXPRESSION OF COFFEE BEAN a-GALACTOSIDASE IN Pichia pastoris

non is probably due to different copy numbers and ar-rangement of the gene incorporated into the P. pastorisgenome (12). In general, transformants with highercopy numbers of the inserted gene tend to produce moreprotein than those with lower copy numbers. Scorer etal. developed a method of using G-148 resistance as aselection marker for screening high copy number trans-formants (13). An exception to the correlation betweenthe copy number and protein production level has beenobserved in the case of D-alanine carboxypeptidase (14);therefore, we developed an assay to directly screen forP. pastoris transformants with high-level expressionof a-galactosidase rather than screening for high copynumbers. The growth and induction of P. pastoris colo-

FIG. 4. Purification of recombinant a-galactosidase produced in P. nies on a nitrocellulose membrane made it possible topastoris. After the culture supernatant was loaded onto the Macro directly assay the activity of a-galactosidase expressedPrep S-50 column and unbound proteins were washed out, the a- in colonies. The intensity of the blue color generatedgalactosidase was eluted from the column with the NaCl gradient

by the enzyme provides the first indication of the ex-and collected in 12-ml fractions. The amount of protein and enzymepression level, although a shake flask culture (secondactivity in different fractions was measured by the absorbancy at

280 nm and the activity assay, respectively. phase screening) is necessary for a more quantitativeanalysis. In addition, the activity assay on the mem-brane can effectively eliminate from further analysisthose colonies which are His/ but enzymatically inac-PAGE and the gel was stained with Coomassie blue. tive. By applying this assay, we were able to selectAs shown in Fig. 5, a single protein band (lanes 5 and a high-expression clone, paF-BZ-22, from P. pastoris6) was visualized and was indistinguishable from the transformed with SalI-digested paF-BZ. The reasonnative enzyme (lane 7). In addition, the purified protein we chose SalI- rather than the BglII-digested plasmidgave a positive Western blot with the antibody raised is not only because the former offered higher transfor-against the native enzyme (data not shown). The spe- mation efficiency but also the transformant thus gener-cific activity for a-galactosidase increased from 12.5 to ated (Mut/ phenotype) is more easily scaled up and32 U/mg following chromatography on the S-50 column, reaches higher cell density under fermentation condi-representing a 2.6-fold increase in purification. tions.The purity of recombinant a-galactosidase after the In comparison with the shake flask procedure, cul-S-50 column was further confirmed by chromatofocus- turing P. pastoris in a fermentor generates signifi-ing and C-4 reverse-phase chromatography (data not cantly higher expression levels. We observed a 10-foldshown). In both experiments, a single peak was ob- increase in a-galactosidase activity per milliliter of cul-served, indicating the homogeneous nature of the pro- ture medium. Nevertheless, the data derived fromtein. In addition, an isoelectric point of 6.2 was ob- shake flask studies are important in order to determinetained by chromatofocusing of the recombinant en- which yeast transformants are higher producers andzyme. Thus, the enzyme produced in P. pastoris has thus best suited for fermentation production. The con-the same characteristics as native enzyme (isoform II)

isolated from green coffee beans (2). N-terminal se-quencing of the purified protein suggested that the sig-nal sequence of a mating factor derived from the plas-mid was cleaved at two positions (site II and III in Fig.1), generating two forms of a-galactosidase with Phe orLeu as the N-terminal residue in approximately equalmolarity. Half of the expressed protein then has theidentical N-terminus (Leu residue) as the mature en-zyme isolated from green coffee beans (6) and the otherhalf has an additional residue, Phe, at the N-terminus,which was coded by the vector sequence (see Fig. 1). FIG. 5. SDS–PAGE stained with Coomassie blue. Different frac-

tions from the S-50 column (see Fig. 4) were analyzed by SDS–PAGE.Lane 1, supernatant of P. pastoris culture; lane 2, unbound fractionDISCUSSIONfrom the column; lane 3, fraction 55; lane 4, fraction 65; lane 5,

A high degree of clonal variation in the level of the fraction 80; lane 6, fraction 150; lane 7, native a-galactosidase; andlane 8, size marker (kDa).expressed protein has been reported and this phenome-


70 ZHU ET AL.

ditions for cell growth and protein induction in a shake digested with CNBr followed by HPLC separation, theHPLC profiles were indistinguishable (unpublishedflask were optimized in terms of two parameters,data). It is not clear what structural difference is re-amount of methanol added and pH of the culture me-sponsible for these two forms of the enzyme anddium. Although a similar level of enzyme induction waswhether such a difference is unique to the source or theobtained by adding either 5% methanol once at theisolation procedure. According to the cDNA sequenceinitiation of induction or 1% methanol daily. We chosethere is one putative N-glycosylation site; however, nothe one-time addition of 5% methanol for its conve-glycosylation was detected in either the recombinant ornience. No adverse effect on the cell growth was ob-native a-galactosidase. Interestingly, the recombinantserved under such conditions. The pH of the cultureenzyme displayed only a single peak by chromatofocus-medium is another important parameter for high-leveling which corresponds to isoform II (2). Further charac-expression of a-galactosidase and must be determinedterization of both forms of a-galactosidase may shedexperimentally in the shake flask as well as in thelight on their structural differences, which in turn mayfermentor. As indicated in Fig. 2B, the optimal pH ofcontribute to the observed distinction in their pI andthe shake flask culture is pH 6.5, whereas the optimaloptimal pH.pH for the fermentor culture ranges from 4 to 5 (Fig.

The data presented here demonstrate that P. pas-3). Such a discrepancy may result from their differencetoris is a powerful tool for large-scale production of re-in growth environment and culture media.combinant a-galactosidase. Further characterization ofAccording to the construction of the expression plas-the recombinant a-galactosidase is in progress and willmid paF-BZ (Fig. 1), the signal sequence of a matingbe reported elsewhere (16). With such an efficient ex-factor was thought to be cleaved after the dibasic resi-pression system and purification procedure, largedues Lys-Arg by the KEX2 gene product (15) and thenamounts of purified recombinant enzyme can be readilyGlu-Ala repeats may be further cleaved by dipeptidyl-prepared for use in the study of B r O blood type con-amino-peptidase encoded by the STE 13 gene (site I inversion.Fig. 1). Thus, the recombinant a-galactosidase may

have an extra four amino acid residues at the N-termi-REFERENCESnus in comparison with the enzyme isolated from coffee1. Dey, P. M., and Pridham, J. B. (1972) Adv. Enzymol. 36, 91–beans. However, N-terminal sequencing of purified re-

130.combinant enzyme indicated that the Phe or Leu was2. Courtois, J. E., and Petek, F. (1966) Methods Enzymol. 3, 565–the first residue at the N-terminus in approximately 571.

equimolar amounts (sites II and III in Fig. 1). Thus, 3. Yagi, F., Eckhardt, E., and Goldstein, I. J. (1990) Arch. Biochem.approximately half of the recombinant a-galactosidase Biophys. 280, 61–67.has the identical N-terminal sequence as the native 4. Harpaz, N., Flowers, H. M., and Sharon, N. (1975) Arch. Bio-

chem. Biophys. 170, 676–683.enzyme and the other half contains an additional Phe5. Goldstein, J., Siviglia, G., Hurst, R., and Lenny, L. (1982) Scienceresidue at the N-terminus. This was unexpected be-

215, 168–170.cause the sequence at cleavage sites II and III are dif-6. Lenny, L. L., Hurst, R., Goldstein, J., and Galbraith, R. A. (1994)ferent from the authentic signal sequence of coffee bean Transfusion 34, 209–214.

a-galactosidase. The mature a-galactosidase sequence 7. Zhu, A., and Goldstein, J. (1994) Gene 140, 227–231.downstream of sites II and III appears to provide a 8. Cregg, J. M., Vedvick, T. S., and Raschke, W. C. (1993) Bio/cleavage signal that the host posttranslational machin- Technology 11, 905–909.ery can recognize. In addition, the folding of the mature 9. Coppola, G., Yan, Y., Hantzopoulos, P., Segura, E., Stroh, J. G.,

and Calhoun, D. H. (1994) Gene 144, 197–203.a-galactosidase may make the N-terminal heterologous10. Ioannou, Y. A., Bishop, D. F., and Desnick, R. J. (1992) J. Cellpeptide sequence more accessible to host protease di-

Biol. 119, 1137–1150.gestion. The data presented here do not rule out the11. Fellinger, A. J., Verbakel, J. M. A., Veale, R. A., Sudbery, P. E.,possibility that the signal sequence of yeast a mating Bom, I. A., Overbeeke, N., and Verrips, C. T. (1991) Yeast 7,

factor is cleaved at site I, which could be an indepen- 463–473.dent event from cleavage at sites II and III. 12. Clare, J. J., Rayment, F. B., Ballantine, S. P., Sreekrishna, K.,

and Romanos, M. A. (1991) Bio/Technology 9, 455–460.In the course of studying coffee bean a-galactosidase13. Scorer, C. A., Clare, J. J., McCombie, W. R., Romanos, M. A.,we have observed two forms of the enzyme with differ-

and Sreekrishna, K. (1994) Bio/Technology 12, 181–184.ent pIs and optimal pHs (Goldstein et al., unpublished14. Despreaux, C. W., and Manning, R. F. (1993) Gene 131, 35–41.data). In order to explore their possible structural dif-15. Brake, A. J., Merryweather, J. P., Coit, D. G., Heberlein, U. A.,ferences, we isolated these two isoforms from green

Masiarz, G. R., Mullenbach, G. T., Urdea, M. S., Valenzuela, P.,coffee beans by preparative chromatofocusing and the and Barr, P. J. (1984) Proc. Natl. Acad. Sci. USA 81, 4642–4646.N-terminal sequencing indicated that both isoforms 16. Zhu, A., Leng, L., Monahan, C., Zhang, Z., Hurst, R., Lenny, L.,

and Goldstein, J., submitted.had identical N-termini. When the two isoforms were


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High-Level Expression and Purification of Coffee Bean α-Galactosidase Produced in the YeastPichia pastoris