Characterization of the kexin-like maturase of Aspergillus niger

Abstract

Secreted yields of foreign proteins may be enhanced in filamentous fungi through the use of translational fusions in which the target protein is fused to an endogenous secreted carrier protein. The fused proteins are usually separated in vivo by cleavage of an engineered Kex2 endoprotease recognition site at the fusion junction. We have cloned the kexin-encoding gene of Aspergillus niger (kexB). We constructed strains that either overexpressed KexB or lacked a functional kexB gene. Kexin-specific activity doubled in membrane-protein fractions of the strain overexpressing KexB. In contrast, no kexin-specific activity was detected in the similar protein fractions of the kexB disruptant. Expression in this loss-of-function strain of a glucoamylase human interleukin-6 fusion protein with an engineered Kex2 dibasic cleavage site at the fusion junction resulted in secretion of unprocessed fusion protein. The results show that KexB is the endoproteolytic proprotein processing enzyme responsible for the processing of (engineered) dibasic cleavage sites in target proteins that are transported through the secretion pathway of A. niger.

FIG. 1

Sequence characteristics of the A. niger kexB gene. (A) Partial restriction map of the kexB genomic region. The position of the ClaI (C), KpnI (K), and SalI (S) used in cloning strategies are indicated. The distance between the KpnI sites flanking the cloned region is approximately 10 kb. The ORF is indicated with a gray box and the arrow indicates the direction of transcription. The position of the single intron in kexB is indicated with a black box. (B) Putative domains of the KexB protease. The KexB amino acid sequence is indicated by an open box. The following domains could be distinguished: signal-sequence (S), prosequence (Pro), subtilisin-like domain (Cat), P domain (P), and transmembrane domain (T). The putative auto cleavage site (with arrowhead) and the putative Golgi retention signal are also indicated.

FIG. 2

Molecular characterization of kexB multicopy strains. (A) Map of the insert of pIM4002 used for transformation. The SalI sites (S) in the 5′ and 3′ untranslated regions of kexB are replaced by KpnI (K) sites. (B) Southern analysis of kexB-overexpressing transformants. KpnI-digested genomic DNA of NW249 and of five transformants was analyzed. The 10-kb band of the endogenous gene and the 4-kb band originating from intact integrated copies of pIM4002 are indicated by arrows. Some scattered integration of pIM4002 is also observed. (C) Northern analysis of kexB expression in multicopy transformants. Transformant numbers are indicated above the lanes. In the lower panel, the membrane is rehybridized with ribosomal protein gene rpS28 to provide a loading control. WT, wild type.

FIG. 3

Molecular characterization of kexB disruption strains. (A) Map of the insert of pIM4003 used for transformation. A part of the kexB ORF substituted with the argB selection marker. The BamHI (B), ClaI (C), EcoRI (E), KpnI (K), HindIII (H), PstI (P), and SalI (S) used in the cloning strategy are indicated. (B) Southern analysis of arginine prototrophic transformants. PstI-digested genomic DNA of NW249 and of 11 transformants was hybridized with a SalI-EcoRI fragment of pIM4003. The endogenous kexB gene hybridizes as a 15-kb fragment (WT lane). A 4.4-kb fragment replaces this fragment if the argB gene replaces a part of the kexB coding region (lanes 3, 4, 8, and 11). Transformants (lanes 1, 6, 7, 9, and 10) show an ectopic pattern of integration. Transformants (lanes 2 and 5) have integrated only a functional argB gene.

FIG. 4

Time dependence of hydrolysis of Boc-Leu-Lys-Arg-MCA by the DSP fractions of NW249 (■), MCK-5 (▴), and NW266 (⧫). The data represent the means of two independent experiments. Standard errors are indicated by bars or are within each symbol.

FIG. 5

Ca²⁺ dependency of KexB activity. Incubations were done for 4 h in 200 mM HEPES, 0.2 mM EDTA (pH 7.0), and a varying Ca²⁺ concentration with 10 μl of DSP extract of NW249 (1.33 mg of protein/ml) and 100 μM Boc-L-K-R-MCA as a substrate. The data represent the means of two independent experiments. Bars indicate standard errors.

FIG. 6

Western analysis of glucoamylase–human interleukin-6 processing. Medium samples (0.5 ml) of MCGI (wild type) (WT) and MCGIΔ (kexB disruptant) (Δ) were analyzed for the presence of unprocessed human interleukin-6 (glaA-KEX2-hIL6). The control lane (C) contains 0.5 μg of recombinant human interleukin-6 (hIL6).