Expression, Purification, and Characterization of Recombinant α-N-Acetylgalactosaminidase Produced in the YeastPichia pastoris

PROTEIN EXPRESSION AND PURIFICATION 8, 456–462 (1996)ARTICLE NO. 0124

Expression, Purification, and Characterization ofRecombinant a-N-AcetylgalactosaminidaseProduced in the Yeast Pichia pastoris

Alex Zhu, Catherine Monahan, Zhong-Kun Wang, and Jack GoldsteinLindsley F. Kimball Research Institute of the New York Blood Center, 310 East 67 Street, New York, New York 10021

Received February 7, 1996, and in revised form August 1, 1996

a-N-Acetylgalactosaminidase (aNAGAL, EC 3.2.1.49),an exoglycosidase specific for terminal a-linked N-acetyl-a-N-Acetylgalactosaminidase (aNAGAL, EC 3.2.1.49)galactosamine (galNAc), has been identified and purifiedpurified from chicken liver has been used in serocon-from a variety of species including bacteria, protozoa,version of human erythrocytes. Blood group A, definedgastropods, and mammalian liver (1–5). The cDNA, how-by the terminal a-linked N-acetylgalactosamine, canever, has only been cloned and sequenced from twobe cleaved in vitro by aNAGAL, resulting in the under-sources, human and chicken liver (6–8). Humanlying penultimate blood group H (O) epitope structure.aNAGAL plays an important role in catabolism of glyco-In order to produce sufficient quantities of recombi-conjugates and its deficiency may cause Schindler diseasenant aNAGAL (raNAGAL) for such studies, we ex-(9). aNAGAL purified from chicken liver has been shownpressed the cDNA encoding chicken liver aNAGAL in

Pichia pastoris, a methylotrophic yeast strain. The to efficiently cleave the terminal a-linked galNAc fromaNAGAL coding sequence was cloned into the EcoRI group A red cells. (10). Since group O erythrocytes aresite of the vector pPIC 9 such that the protein was in considered ‘‘universal donors,’’ the enzymatic conversionthe same reading frame as the secretion signal of yeast of red cells from blood group A to O would be very im-a-mating factor derived from the vector. After P. pas- portant for transfusion medicine (11). Unfortunately, pu-toris transformation, colonies were screened for high- rification of chicken liver aNAGAL is both labor-intensivelevel expression of raNAGAL based on enzyme activ- and time-consuming (12). Recently, we reported the mo-ity. As a result of methanol induction of high-density lecular cloning of chicken liver aNAGAL cDNA by librarycell cultures in a fermentor, enzymatically active screening and PCR amplification (13). In this reportraNAGAL was produced and secreted into the culture chicken liver aNAGAL is expressed in Pichia pastoris,medium. The recombinant enzyme was purified over a methylotrophic yeast strain, and the purification and150-fold by chromatography on a cation exchange col- characterization of the recombinant enzyme are de-umn followed by an affinity column. Its homogeneity scribed.was confirmed by Coomassie blue-stained SDS–PAGE,Western blot, and N-terminal sequencing. The purifiedraNAGAL has a molecular mass of approximately 50 METHODSkDa while its native counterpart has a molecular massof 43 kDa. This discrepancy in size was eliminated by Cell Lines and Culture Mediaendoglycosidase treatment, suggesting that the recom-

DNA subcloning was completed in E. coli JM109 cellsbinant protein was hyperglycosylated by the host P.(Promega) while yeast transformations and protein ex-pastoris cells. raNAGAL was further characterized inpression were done in P. pastoris GS115 (his40) (In-terms of specific activity, pH profile, kinetic parame-

ters, and thermostability by comparing with aNAGAL vitrogen). The media components for P. pastoris trans-purified from chicken liver. The data presented here formation and cell growth are described by Invitrogensuggest that by overexpressing raNAGAL in P. pastoris and include: regeneration dextrose base and top agarand purifying with affinity chromatography one can (RDB), minimal dextrose plates (MD), minimal metha-readily obtain the quantity of enzyme needed for sero- nol plates (MM), buffered minimal glycerol-complexconversion studies. q 1996 Academic Press, Inc. media (BMGY), and YPD. The components for fermen-

tation culture have been listed previously (14).

456 1046-5928/96 $18.00Copyright q 1996 by Academic Press, Inc.

All rights of reproduction in any form reserved.

AID PEP 0649 / 6q0a$$$341 10-31-96 14:30:26 pepa AP: PEP

EXPRESSION OF a-N-ACETYLGALACTOSAMINIDASE IN Pichia pastoris 457

FIG. 1. The 5* cloning site of the plasmid paF-AZ and N-terminus of raNAGAL produced in P. pastoris. EcoRI site of the vector pPIC9was used for the cloning of aNAGAL cDNA. The shaded sequence is part of the signal peptide of yeast a mating factor and the boxedpeptide indicates the beginning of mature aNAGAL isolated from chicken liver. Arrows indicate the multiple N-termini of raNAGALsecreted into P. pastoris culture medium. The quantity of the molecules with different N-termini decreased in the order (a, b, c, d).

Plasmid Construction and P. pastoris Transformation clone was inoculated in 5 ml BMGY and grown at 307Cand 300 rpm until the cell density reached 5 1 108

The cDNA encoding mature chicken liver aNAGALcells per milliliter. The cultures were then induced bywas cloned into the EcoRI site of both P. pastoris ex-adding 5% methanol directly to the BMGY culture andpression vectors pPIC9 and pHIL-S1 (Invitrogen), gen-grown at 307C and 300 rpm for 4 days. The cultureserating paF-AZ and pHO-AZ, respectively. The cDNAwere centrifuged at 14,000 rpm for 5 min, the pellet ofwas in the same open reading frame as the upstreamcells was discarded, and the supernatant was retainedsecretion signal which is derived from a-mating factorand tested directly for aNAGAL activity. The highestin paF-AZ and derived from the PHO1 gene product inactivity levels were exhibited by the pHO-AZ-23 trans-pHO-AZ. The 5* cloning site of the plasmid paF-AZ isformant at 0.044 U/ml and by the paF-AZ-84 trans-illustrated in Fig. 1. Yeast transformations were doneformant at 0.069 U/ml; thus, these two colonies wereusing the spheroplast method detailed by Invitrogenchosen for raNAGAL production by fermentation.with 1.2 mg of pHO-AZ and 1.5 mg of paF-AZ, linearized

with SalI.Large-Scale Production and Protein Purification

Functional Selection of Transformants Expressing High-density cell culture was carried out in a 14-literaNAGAL module MicroFerm bench-top fermentor MF-114 (New

Brunswick Scientific Co., Inc.) following standardizedYeast transformants were analyzed for aNAGAL ac-tivity by a two-phase process consisting of an initial conditions (14). In the first phase of fermentation, the

cell density reached about 200 g (wet weight) of cellsmembrane screening procedure followed by growth andexpression in a culture assay as described by Zhu et per liter of culture after feeding glycerol for 2 days. In

the second phase, methanol was fed at a rate of 3 ml/al. (14). The first phase of the screening process wascompleted by making a nitrocellulose replica lift of the h/liter for raNAGAL induction. In about 5 days the

level of raNAGAL secreted into the culture mediumcolonies on the surface of the RDB top agar of the trans-formation plates. The colonies were grown and induced reached a plateau. Throughout the fermentation run,

the cell culture was maintained under the followingdirectly on the membrane by placing the lift first onMD plates at 307C for 7–16 h and then transferring conditions: 307C, pH 5–5.5, 20% of condensed air flow,

and 1000 rpm of agitation.the lift to MM plates at 307C for 16 h. The lift was thenexposed to 5-bromo-4-chloro-3-indolyl-a-D-acetamido- After the level of raNAGAL reached a plateau, P.

pastoris cells were removed by centrifugation at 35002-deoxy-galactopyranoside (X-a-galNAc, 1 mg/ml in ci-trate buffer, pH 3.6). Of the total colonies exhibiting a rpm for 30 min. Supernatant containing raNAGAL was

concentrated approximately 10-fold and equilibratedyellow to blue color change, indicating positiveaNAGAL activity for the first-phase membrane screen- with 20 mM sodium acetate buffer, pH 4.5, using a

10,000 MW cutoff ultrafiltration membrane (Milliporeing protocol, 7 pHO-AZ and 50 paF-AZ transformantswere chosen for the second-phase culture assay. Each Corp.). After removing insolubles by centrifugation at


ZHU ET AL.458

raNAGAL was then dialyzed extensively against ci-trate buffer and stored at 0207C for future use.

Physico-chemical Characterization of raNAGAL

Protein concentration was determined by BCA pro-tein assay (Pierce Chemicals) using bovine serum albu-min as the protein standard. raNAGAL activity wasmeasured by incubating the sample with 2.5 mM of p-nitrophenol-a-N-acetylgalactosaminoside (PNP-a-gal-NAc) at pH 3.65 and 377C. The reaction was terminatedby adding 0.2 M borate buffer, pH 9.8, and then theabsorbency of the reaction was measured at 405 nm.One unit (U) of activity is defined as the amount ofenzyme required to hydrolyze 1 mmol of substrate perminute under the assay conditions. Kinetic parameters(Km and Vmax) were calculated from initial velocitiesin the 0.2–5.0 mM range of PNP-a-galNAc with thecomputer program Enzypack 3 (Biosoft). Protein sam-ples were analyzed on SDS–acrylamide gels (precastfrom Bio-Rad) and the Western blots were carried outusing ProtoBlot system (Promega) with a polyclonalantibody raised against aNAGAL purified from chickenliver. Deglycosylation was carried out by denaturingprotein samples in 1% SDS, 1007C for 2 min prior tothe endoglycosidase treatment (incubated at 377C for1 h with N-glycosidase F from Boehringer Mannheimor endoglycosidase H from Genencor).

FIG. 2. Column chromatography of recombinant aNAGAL pro-RESULTSduced in P. pastoris. (A) aNAGAL activity was eluted from the Macro-

Prep S-50 column by a NaCl gradient (dashed line) ranging from 50 Expression of aNAGAL cDNA in P. pastoristo 500 mM. Column fractions (12 ml each) were measured for protein(OD280) and were assayed for aNAGAL activity toward PNP-a-gal- In order to compare the production level in P. pas-NAc (OD405). The fractions containing raNAGAL, as indicated by toris, cDNA encoding chicken liver aNAGAL wasthe back-slashed bar, were pooled for the affinity column of N-e-

cloned into EcoRI site of both expression vectors, pPICaminocaproyl-a-D-galactopyranosylamino-Sepharose. After washing9 and pHIL-SI. Although both vectors use the metha-off the unbound material, raNAGAL was specifically eluted from the

column with 0.05 M a-galNAc (B). Fractions 3 to 7 were combined nol-inducible promoter of the alcohol oxidase genefor protein characterization. (AOX), they contain different signal sequences located

downstream from the promoter for the secretion of ex-pressed proteins. The signal sequence in pPIC 9 is de-rived from the Saccharomyces cerevisiae a-mating fac-13,000g for 30 min, the sample was applied to a 50-

ml cation-exchange column MacroPrepS-50 (Bio-Rad tor, whereas, the signal sequence in pHIL-S1 derivedfrom the acid phosphatase gene, PHO1. Laroche et al.Laboratories) equilibrated with 20 mM NaAc buffer, pH

4.5. The column was washed extensively with the NaAc (15) reported a higher level of expression of tick antico-agulant peptide in P. pastoris using the pPIC 9 vectorbuffer containing 50 mM NaCl followed by a linear

NaCl gradient from 50 to 500 mM. The column fractions rather than the pHIL-SI vector. On the other hand, nodiscrepancy was observed in the level of expression,(12 ml each) were measured at 280 nm for protein and

assayed for raNAGAL activity. Fractions containing secretion efficiency, and enzyme activity between thetwo vectors producing raNAGAL in P. pastoris. Thus,raNAGAL were pooled and loaded onto a second col-

umn containing e-aminocaproyl-aD-galactopyranosy- all data presented below were obtained from paF-AZ-transformed P. pastoris.lamine-Sepharose (Sigma) preequilibrated with 50 mM

sodium citrate buffer (pH 4.5). The column was washed Pichia pastoris transformation can be accomplishedwith plasmid DNA linearized with either SalI or BglIIwith sodium citrate buffer followed by buffer con-

taining 0.25 M NaCl. Finally, raNAGAL activity was for gene integration at the HIS 4 locus or AOX1 locus,respectively. Previous studies of recombinant a-galac-eluted from the column with citrate buffer containing

0.05 M of N-acetyl-a-galactosamine. The purified tosidase indicated that higher transformation effi-



TABLE 1

Purification of aNAGAL Produced in P. pastoris

Volume Protein Activity Specific activity Purification Overall yieldStep (ml) (mg) (Units) (U/mg) (fold) (%)

Culture supernatant 10,090 37,333 13,312 0.36 1.0 100.0Concentration 950 7,230 10,346 1.4 4.0 77.7S-50 column 595 1,261 6,854 5.4 15.2 51.5Affinity column 63 117 6,535 55.9 156.6 49.1

Note. The data were derived from approximately 10 liters of P. pastoris culture supernatant at the end of a 5-day methanol inductionperiod.

ciency was observed with SalI-digested plasmid (14); rpm for 30 min, approximately 10 liters of the superna-tant was obtained. The binding capacity of the Macro-therefore, 1.5 mg of paF-AZ DNA was linearized with

SalI prior to P. pastoris transformation. A total of ap- Prep S-50 resin for raNAGAL under the experimentalconditions is approximately 400 U/ml of the resin.proximately 150 colonies generated were screened for

enzyme expression in the first-phase screening. The Therefore, we applied the culture supernatant con-taining 13,000 U of raNAGAL to a 50-ml S-50 column50 colonies which demonstrated the most intense blue

color were chosen for the second phase of the screening to assure the complete binding of the enzyme. Uponapplying a salt gradient to the column, raNAGAL wasprocess. Finally, the clone with highest level of

raNAGAL expression, paF-AZ-84 (His/Mut/), was cho- eluted when the concentration of NaCl reached morethan 0.1 M, as shown in Fig. 2A. The fractions con-sen for large-scale enzyme production in fermentation

culture. taining the enzyme activity (fractions 15 to 58) werepooled and subjected to the affinity column. After wash-Although P. pastoris cells can grow in a wide variety

of pHs ranging from 3 to 7 in a fermentor, the level of ing unbound proteins off the column, raNAGAL (frac-tions 3 to 7) was eluted with 0.05 M of the specificexpressed protein is affected by the pH of the culture.

At the extreme ends of this pH range, raNAGAL is not monosaccharide, as shown in Fig. 2B.As summarized in Table 1, this purification proce-stable due to both the nature of the enzyme and the

activation of endogenous proteases. The culture me- dure produced over 6500 U (116 mg) of purifiedraNAGAL from the 10-liter culture supernatant withdium was maintained at pH 5–5.5 during cell growth

and enzyme induction in the fermentor in order for the 157-fold purification and 49% overall yield. However,recovery of the enzyme activity from the S-50 columnexpressed raNAGAL to remain stable. After the cell

density reached as high as 220 mg/ml of culture, meth- was only 66% when compared with the recovery withthe affinity column at 95%. In order to increase theanol feeding at 3 ml/h was initiated. The level of raNA-

GAL secreted into the culture medium reached a pla- overall yield and simplify the procedure, the concen-trated supernatant was applied directly to the affinityteau (1–1.2 U/ml) after 5 days of induction.

It is worthwhile pointing out that, under essentially column. Following the same conditions describedabove, raNAGAL was eluted from the column andthe same conditions for cell growth and induction in the

fermentor, recombinant a-galactosidase was secreted found to migrate as a single band on Coomassie blue-stained SDS–PAGE, (see Fig. 3A, lane 8).into the culture medium and reached a level as high

as 12 U/ml (14). It is unclear why there is such a dis-crepancy in the level of secreted proteins between these Characterization of Purified raNAGALtwo enzymes. However, by examining the expressedproteins accumulated inside the cells in a Western Samples for each step of purification were loadedblot, we detected substantially greater amounts of onto a 0.1% SDS–12% polyacryamide gel, subjected toraNAGAL than recombinant a-galactosidase (data not electrophoresis followed by Coomassie blue staining.shown). Thus, it seems that the low production level of As shown in Fig. 3A, combined fractions from the gradi-raNAGAL in the culture media may result, at least ent elution of the S-50 column contained raNAGALpartially, from its low secretion efficiency. and several low-molecular-weight (MW) contaminants

(lane 4). However, if only the fractions correspondingPurification of raNAGAL Produced in P. pastoris to the peak of enzyme activity (refer to Fig. 2A) were

analyzed by SDS–PAGE, those low-MW bands wereInduced P. pastoris fermentation culture (12 liters)was collected at the end of the 5-day induction period apparently absent (data not shown). Nevertheless, we

combined all the fractions containing raNAGAL in or-and contained approximately 310 g of wet weight cellsper liter of the culture. After centrifugation at 3500 der to increase overall recovery of the enzyme. Since


ZHU ET AL.460

fucose residues present in insect and plant glycopro-teins, whereas endoglycosidase H cleaves only high-mannose structures. As shown in Fig. 3B, after treat-ment with endoglycosidase H (lane 2) or N-glycosidaseF (lane 3), raNAGAL migrated to a position slightlylower than its native counterpart (lane 4). On the otherhand, the reduction in molecular mass of naNAGALupon deglycosylation was more pronounced with N-gly-cosidase F (lane 6) than with endoglycosidase H (lane5). The data suggest that raNAGAL produced inP. pastoris is hyperglycosylated and differs fromnaNAGAL in the nature of glycosylation. Similar ob-servations have been made in studies of expression ofHIV-1 envelope protein and human gastric cathepsinE in P. pastoris (16,17). As indicated earlier, we ob-served low secretion efficiency of raNAGAL. This maybe due to the hyperglycosylation of the recombinantprotein. Currently, we are testing this hypothesis byaltering putative N-glycosylation sites in aNAGALcDNA using site-directed mutagenesis. In addition, thepurified raNAGAL contained some high-MW smear asindicated in Coomassie blue-stained SDS–PAGE(lanes 4, 6, and 8 of Fig. 3A). However, its treatment

FIG. 3. Purification of raNAGAL and treatment with endoglycosi- with endoglycosidase H resulted in a single band ofdases. (A) Samples from each step of purification scheme shown in reduced size on SDS–PAGE (Fig. 3C), suggesting thatTable 1 were analyzed by SDS–PAGE stained with Coomassie blue. the high-MW smear may represent a small portion ofLane 1, molecular mass standards; lane 2, culture supernatant (20

raNAGAL containing extensive hyperglycosylation.ml); lane 3, concentrated supernatant (10 ml); lane 4, combined frac-tions from the S-50 column (10 ml); lane 5, unbound material from Since aNAGAL cDNA was cloned, in the same read-the affinity column (10 ml); and lane 6, eluate from the affinity column ing frame, downstream from a secretion signal,(2 ml). In a separate experiment, concentrated culture supernatant raNAGAL was secreted into the culture medium afterwas applied directly to the affinity column and the eluate (6 ml) is the signal peptide was cleaved in the host cells. N-shown in lane 8. Lane 7 contains 2.5 mg of aNAGAL purified from

terminal sequencing of purified aNAGAL revealedchicken liver as a control. The numbers to the left of lane 1 indicatethe molecular mass in kDa (Bio-Rad Laboratories). (B) Protein sam- multiple termini as indicated in Fig. 1. Based on theples were treated with endoglycosidases and then examined by West- amount of residues released in each cycle of proteinern blotting. Lanes 1 and 4 are untreated raNAGAL and naNAGAL sequencing, the percentage of the aNAGAL moleculescontrols, respectively. Lanes 2 and 3 are raNAGAL treated with

with different N-termini decreased in the order (a, b,endoglycosidase H and N-glycosidase F, respectively. Lane 5 and 6c, d) shown in Fig. 1. Cleavage at site ‘‘a’’ may resultare naNAGAL treated with endoglycosidase H and N-glycosidase F,

respectively. (C) raNAGAL was treated with endoglycosidase H and from the KGX 2 gene product and then Glu–Ala re-examined by SDS–PAGE stained with Coomassie blue. Lanes 1 and peats may be further cleaved to generate sites ‘‘c’’ and2 are treated and untreated raNAGAL samples, respectively. ‘‘d’’ by dipeptidylaminopeptidase encoded by the STE

13 gene. It is not clear which protease was responsiblefor cleavage at ‘‘b.’’ However, it is worth pointing outthat the same cleavage at site ‘‘b’’ was observed in athe low-MW protein contaminants were readily washed

from the affinity column (lane 5), the eluant (lane 6) similar construct for the expression of recombinant a-galactosidase in P. pastoris (14).apparently contained only raNAGAL. Based on the size

marker (lane 1), the molecular mass of purified Specific activity, optimal pH, and kinetic parametersof raNAGAL were compared with those of naNAGALraNAGAL is approximately 50 kDa, whereas its native

counterpart (naNAGAL) has a molecular mass of 43 and are summarized in Table 2. Conducting the en-zyme assay at pH values ranging from 2.0 to 8.0, thekDa (lane 7). In order to determine whether such a

discrepancy in molecular weight was caused by over- highest activity of raNAGAL with PNP-agalNAc sub-strate was observed at pH 3.6, which is identical toglycosylation by the host cells P. pastoris, the purified

raNAGAL, as well as its native counterpart, was that of naNAGAL. Although the specific activity ofraNAGAL (51.2 U/mg) is slightly lower than that oftreated with N-glycosidase F or endoglycosidase H and

the results were analyzed by Western blot. N-Glycosi- naNAGAL (56.4 U/mg), the decrease is negligible dueto the difference in molecular weight. While thedase F hydrolyzes all types of N-glycan chains from

glycoproteins except those containing a-1-2-linked core observed differences in the kinetic parameters of



TABLE 2

Comparison of Recombinant and Native aNAGAL

Enzyme Specific activity (U/mg) Optimal pH Vmax (U/mg) Km (mM) Vmax / Km

raNAGAL 51.2 3.65 60.9 0.827 73.6naNAGAL 56.4 3.65 75.7 0.798 94.9

Note. Apparent Michaelis constant (Km) and maximal velocity (Vmax) were generated by the computer program Enzpack 3 based on theLineweaver–Burk method and were expressed in mM and U/mg, respectively.

raNAGAL and naNAGAL should not be affected by 5 to 8. However, when the pH was lower than 5, activityof raNAGAL dropped much more dramatically. Sincemolecular weight, their cause has yet to be understood.

The effect of pH and temperature on raNAGAL was proteases which can be activated at a low pH have beenidentified in P. pastoris (18), one of the possibilities forstudied in comparison with naNAGAL. As shown in

Fig. 4A, both enzymes were stable at a pH range from observed loss of activity at low pH is protease contami-nation in the raNAGAL preparation. An aliquot of puri-fied raNAGAL was adjusted to pH 3.5 and incubatedfor 5 h before being subjected to a Western blot usingthe antisera specific for aNAGAL. The protein re-mained intact and no smaller bands were observed(data not shown), thus excluding significant proteolyticdegradation. In addition, the temperature–stabilitystudies indicated that although both the native andrecombinant enzymes were equally thermostable up to457C, raNAGAL became much more unstable at a tem-perature higher than 457C (Fig. 4B). It is still unknownwhether the hyperglycosylation of raNAGAL producedin P. pastoris contributes to the apparent discrepancyobserved in the study of pH and temperature stability.It would be informative to carry out these studies withendoglycosidase-treated raNAGAL. Currently, we aretesting the endoglycosidase treatment under nondena-turing conditions so that the activity of deglycosylatedraNAGAL can be measured. Nevertheless, raNAGALproduced in P. pastoris demonstrates similar proper-ties as its native counterpart at 377C and pH 6, whichare conditions we use in the study of blood group ArOseroconversion. Therefore, we plan to use purifiedraNAGAL in the treatment of human red cells andcharacterize the serological and immunological proper-ties of those treated cells.

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Documents

Expression, Purification, and Characterization of Recombinant α-N-Acetylgalactosaminidase Produced in the YeastPichia pastoris