APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/99/$04.0010

May 1999, p. 2250–2252 Vol. 65, No. 5

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

A Sandwiched-Culture Technique for Evaluation ofHeterologous Protein Production in a Filamentous Fungus

SIGRIDUR A. ASGEIRSDOTTIR,* KARIN SCHOLTMEIJER, AND JOSEPH G. H. WESSELS

Molecular Plant Biology Laboratory, Biomolecular Sciences and Biotechnology Institute, University of Groningen,9751 NN Haren, The Netherlands

Received 12 November 1998/Accepted 1 March 1999

Aspergillus niger is known for its efficient excretion machinery. However, problems have often arisen inobtaining high amounts of heterologous proteins in the culture medium. Here we present a quick method usingsandwiched colonies to evaluate transgenic strains for secretion of heterologous proteins. Expressing the ABH1hydrophobin of Agaricus bisporus in A. niger, we showed that low production levels of the heterologous proteinare probably due to extracellular proteolytic degradation of the protein.

We have initiated heterologous expression studies of hydro-phobin genes in Aspergillus niger. For these studies, we havechosen the ABH1 (hypA) gene, which encodes a class I hydro-phobin expressed in the fruiting bodies of Agaricus bisporus,the white button mushroom (2, 3). Because of the character-istic self-assembly of these hydrophobins into sodium dodecylsulfate (SDS)-insoluble amphipathic membranes, interest hasarisen in possible medical and industrial applications (12),requiring an efficient method for production of hydrophobins.

An A. niger strain, AB4.1 (cspA1 pyrG1) (10), was trans-formed according to the method described in reference 8 withtwo types of constructs (Fig. 1), both containing the Escherichiacoli hygromycin B phosphotransferase gene (7) allowing forselection of transformants: (i) pAN/ABH1HygB, a cDNA ofABH1, including nucleotides encoding the signal sequence forsecretion (2, 3) cloned behind the gpd promoter and in front ofthe trpC terminator of Aspergillus nidulans (TAN1 transfor-mants), and (ii) pKXABH1/pAN56-2, a cDNA of ABH1 en-coding the mature hydrophobin fused in frame with a trun-cated A. niger glucoamylase gene (glaA-G2), separated by aKEX2 protease site and cloned between the gla promoter of A.niger and the trpC terminator of A. nidulans (TAN2 transfor-mants). Sequence analysis of the ABH1 cDNA, including thecloning sites, verified the correctness of both constructs.

Our initial experiments analyzed the presence of ABH1 inthe medium of 2-day-old shaken cultures of 10 TAN1 and 30TAN2 transformants. The cultures were grown in minimalmedium (13) at 150 rpm and 30°C with 5% maltodextrin re-placing glucose for induction of the glucoamylase promoter inthe case of the TAN2 transformants. ABH1 was detected bysilver staining (5) and Western blot analysis (3) in 70% of bothtypes of transformants analyzed, but at low concentrations. Noless than the equivalent of 5 ml of medium per lane wasnecessary in an SDS-polyacrylamide gel electrophoresis(PAGE) gel to obtain a clear ABH1 band. Time course exper-iments showed the same low ABH1 protein level over a cultureperiod of 5 days.

The reason for a low amount of a heterologous protein inthe culture medium may be (i) low expression at RNA and/orprotein level, (ii) malfunction of the secretory machinery, or

(iii) instability of the protein in the culture medium. RNAanalysis (exemplified for two individual TAN1 transformants[Fig. 2]) verified that the production of ABH1 was not ham-pered by a low expression level of the gene, locating the prob-lem at a posttranscriptional level. To discriminate between theremaining possibilities, we used the so-called sandwiched-cul-ture technique (Fig. 3a) previously described (13). A proteinbinding membrane (such as polyvinylidene difluoride [PVDF])immobilizes proteins directly after secretion when placed un-der the colony. Specific antibodies allow for a detection of thenewly secreted proteins.

A perforated polycarbonate membrane (diameter, 4.5 cm;pore size, 0.1 mm) was put onto the surface of 10 ml of per-fectly level solidified minimal medium (13) containing 5% mal-todextrin. The plate was heated to 70°C, and 1 ml of melted1.25% agarose was poured over the surface. The temperatureof the plate was brought to room temperature, and 5 ml ofspore suspension (7 3 107 spores ml21) was added onto thecenter of the plate and allowed to soak into the agarose. Asecond perforated polycarbonate membrane was put on top ofthe agarose layer. In all cases, care was taken that no airbubbles were trapped. The sandwiched colonies of a wild-typestrain (AB4.1) and a TAN1 and a TAN2 transformant wereincubated for 3 days at 30°C, transferred onto PVDF mem-branes overlying fresh agar medium, and incubated for 1 h at30°C. The immunosignals of ABH1 on the PVDF membranes(Fig. 3b) showed predominant secretion at the edge of thecolonies, indicating active secretion in a growing zone of thecolony (as shown previously for glucoamylase [13]) and a clearelevation of ABH1 secretion in a TAN2 transformant com-pared to that in a TAN1 transformant. No reaction was de-tected in the wild type. Therefore, we concluded that theTAN2 construct did result in a high level of ABH1 and effec-tive secretion. Repeated experiments showed identical resultswhen colonies were incubated in the presence of PVDF mem-branes for 15 min and 4, 24, and 48 h.

Proteins are tightly bound to PVDF membranes, but a smallpart of ABH1 (no decrease in immunosignal was detected onthe PVDF membranes after extraction) could be dissociatedfrom the membrane by an incubation with 100% trifluoroaceticacid (TFA) for 30 min at room temperature. After removal ofTFA by an N2 stream, the proteins were taken up in 1 ml ofSDS sample buffer, and 25 ml was subjected to SDS-PAGE andWestern blot analysis (Fig. 4, lane 1). ABH1 antibodies reactedwith a protein band running at a 14-kDa position, representingmonomeric ABH1 (3) successfully cleaved from glucoamylase

* Corresponding author. Present address: Department of Pharma-cokinetics and Drug Delivery, University Center for Pharmacy, Uni-versity of Groningen, A. Deusinglaan 1, 9713 AV Groningen, TheNetherlands. Phone: 31-50-3633292. Fax: 31-50-3633247. E-mail:s.asgeirsdottir@farm.rug.nl.

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at the KEX2 protease site. A 10-times dilution of the samplestill gave a positive signal with anti-ABH1 antibodies (data notshown).

To analyze whether the enhanced excretion of ABH1 insandwiched cultures compared to shaken liquid cultures wasdue to different culture conditions, a control experiment wascarried out. A sandwiched TAN2 colony was grown for 3 daysas described for the previous experiment, transferred to thesurface of 10 ml of static liquid medium, and incubated for 24 hat 30°C. The medium was harvested, and a sample of 25 ml wassubjected to SDS-PAGE. As in shaken cultures, no ABH1 wasdetected in the surface-grown liquid culture (lane 2). Also, noABH1 was detected when the cultures were grown on 1 mlinstead of 10 ml of medium nor when the 10 ml of liquidmedium was concentrated 8 times prior to gel electrophoresis(data not shown). This suggested to us that ABH1 was beingdegraded both in static and in shaken cultures and that deg-radation could be diminished by immobilizing the proteins

(i.e., both ABH1 and proteases) on a PVDF membrane im-mediately after secretion.

We analyzed the stability of ABH1 after dissociating it fromthe PVDF membrane. After incubation for 24 h at 30°C withthe culture medium of a 3-day-old TAN2 transformant, ABH1was not detected (lane 3), whereas its level was not reducedafter incubation in H2O (lane 4). Therefore, it is probable that

FIG. 1. Constructs pAN/ABH1HygB and pKXABH1/pAN56-2. (a) ThecDNA of ABH1, including nucleotides encoding the signal sequence for secre-tion (2, 3) cloned into pAN52-1 NotI (9) behind the gpd promoter and in frontof the trpC terminator of A. nidulans (TAN1 transformants). (b) The cDNA ofABH1 encoding the mature protein, fused in frame with a truncated A. nigerglucoamylase gene (glaA-G2), separated by a KEX2 protease site and cloned intopAN56-2 (6) between the gla promoter of A. niger and the trpC terminator of A.nidulans (TAN2 transformants).

FIG. 2. Northern blot analysis. Lanes 1 and 2, RNA from two randomlychosen TAN1 transformants of A. niger. Lane 3, RNA from fruiting bodies of A.bisporus (strain HorstU1R). Lane 4, wild-type A. niger AB4.1. The upper panelshows hybridization signals with ABH1 cDNA probe; the lower panel shows anethidium bromide staining of rRNA for loading control.

FIG. 3. Sandwiched-culture technique. (a) Schematic view of a sandwichedculture. (b) Immunodetection of secreted ABH1 on PVDF membranes under-lying sandwiched colonies of TAN1 transformant, TAN2 transformant, and wildtype (WT), respectively, for 1 h.

FIG. 4. Western blot analysis of extracellular ABH1 produced in sandwichedcolonies of TAN2 transformants. Lane 1, ABH1 dissociated from PVDF mem-brane by TFA incubation. Lane 2, no ABH1 was detected in static liquid me-dium. Lane 3, ABH1, dissociated from PVDF as in lane 1, was not detectableafter 24 h of incubation at 30°C in a 3-day-old TAN2 culture medium. Lane 4,ABH1, dissociated from PVDF as in lane 1, was intact after 24 h of incubationat 30°C in H2O. Lane 5, partially increased stability of ABH1 was obtained whenthe pH and medium components were adjusted.

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the low amount of ABH1 in the culture medium of the trans-formed strains is due to extracellular proteolysis.

The A. niger strain (AB4.1) used as a recipient for transfor-mation experiments is known to contain extracellular pro-teases, most notably aspergillopepsin A and aspergillopepsinB, both belonging to the class of aspartyl proteases (1, 4).However, the use of a protease-deficient strain, AB1.18 (UVmutated in the pepA gene, encoding aspergillopepsin A) (4),did not yield more secreted ABH1 (data not shown). There-fore, other proteases must be responsible for the degradationof ABH1. Van Noort et al. (11) demonstrated a single pHoptimum for proteolytic activity of A. niger AB4.1 at pH 4.0,whereas no myoglobin degradation was detected at pH 6.5 orhigher. We analyzed the influence of increased pH on theproteolysis of ABH1. The initial pH was increased from 6.0 to7.0, and the buffer capacity of the medium was increased byraising the phosphate concentration to four times the amountindicated by Wosten et al. (13) and adding 2% CaCO3 to themedium. This resulted in a final pH of the medium of 6.1instead of pH 3 to 4 after 24 h of growth of the TAN2 trans-formant. The adjustment of the pH and medium componentsonly partially restored the yield of ABH1 (lane 5, 25-ml sampleof static culture medium). We concluded that efficient expres-sion of ABH1 and some other heterologous proteins in A. nigerawaits a protease-free strain.

We have also found the sandwiched-culture technique areliable method to detect the production of engineered pro-teins which could not be detected in a culture medium ofSchizophyllum commune. This method represents a rapid wayto assess expression levels of a heterologous protein in fila-mentous fungi without interference by protein degradation.

We thank Peter Punt and Roy Montijn of TNO, The Netherlands,for their help and valuable discussions.

S.A.A. was supported by a grant from the Hercules European Re-search Center BV, The Netherlands, and K.S. was supported by theFoundation of Life Sciences, The Netherlands (SLW).

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