Short communicationUsing signal peptide prediction with caution, a case study in Aspergillus niger xylanase
Highlights
► The A. niger XynB signal peptide is predicted to be 1–19 residue. ► XynΔ25 and XynΔ19 were constructed by deleting the 1–19 or 1–25 residues. ► XynΔ25 had 10 °C higher Topt and 21.6-times higher thermostability than XynΔ19. ► XynB real signal peptide is 1–25 and not predicted 1–19 residue. ► Signal peptide prediction should be used with caution.
Introduction
Signal peptide (SP) usually sits at protein N-terminus and plays an important role in its maturation and translocation (http://en.wikipedia.org/wiki/G%C3%BCnter_Blobel), such as, secretion from cytoplasm to cell culture medium, transportation to Glogi apparatus or to endoplasmic reticulum. A database is built for better understanding of how SP functions [1].
In investigation of protein function, E. coli is easy and often used as host cells [2], [3], [4]. When a protein, especially, an eukaryotic one, is expressed in E. coli, the host cell usually cannot identify and delete the protein SP. It has to be deleted artificially through PCR; otherwise, SP would interfere with, or even worse, damage the protein function. A specific SP is usually selected and introduced to secret a recombinant protein from host cell. For example, the SP of Aureobasidium pullulans xylanase was introduced to secret the Penicillum citrinum xylanase into E. coli cell culture medium [2]. As for expression vectors in Pichia pastoris cell, the Saccharomyces cerevisiae α-mating factor is the generally used secretion signal [5]. Because of importance in analyzing protein function, SP is predicted by many webs [1], such as, signalP (http://www.cbs.dtu.dk/services/SignalP), SIG-Pred: Signal peptide prediction (http://www.bioinformatics.leeds.ac.uk/prot_analysis/Signal.html). These computational tools facilitate the investigation of protein function. However, the prediction result is usually not known whether correct or not.
Xylanase (EC 3.2.1.8) is important in biomass conversion, renewable energy production, feed stock, etc. [6], [7], [8]. Because of widely used in producing enzymes for fermentation industry, the Aspergillus niger CBS 513.88 genome was sequenced and annotated [9]. Based on the genome annotation, we cloned an A. niger xylanase (XynB). The enzyme belongs to GH10 family hydrolase. With an intention to investigate its property using E. coli cell, the SP should be identified and deleted artificially through PCR. According to the annotation (Swiss-prot: A2QFV7), the XynB SP was predicted to be the 1–19 residues (Met1–Ser19) [9]. The Penicillium simplicissimum xylanase (Swiss-prot: P56588) is another GH10 family enzyme; however, its SP is determined to be the 1–25 residues [10]. The two enzyme SP residues should be similar, because the XynB and the P. simplicissimum xylanase have 85.8% sequence identity. However, the predicted 1–19 (Met1–Ser19) residue is contrary to the probable 1–25 (Met1–Arg25) residue. With an intention to investigate which one is the real SP, two types of XynB, XynΔ19 and XynΔ25, are constructed by respectively deleting the first 19 or 25 residues. By determining and comparing properties of the two constructs, the 1–25 residue is proved to be the XynB real SP.
Section snippets
Materials and methods
According to the A. niger genome annotation [9], the A. niger xylanase (XynB) gene was cloned into pET20b(+) plasmids (Novagen, Shanghai, China). Two types of XynB, XynΔ19 and XynΔ25, were constructed by respectively deleting the 19 residues (Met1–Ser19) (M1VQIKVAALAMLFASQVLS19) or 25 residues (Met1–Arg25) (M1VQIKVAALAMLFASQVLS19E20P21I22E23P24R25) (Fig. 1). Molecular biology reagents, including Pfu polymerase, restriction endo-nucleases NdeI and XhoI, T4 DNA ligase, DNA marker, protein marker,
Construction of two types of xylanases
The amplified XynΔ19 and XynΔ25 genes appeared on the 1.4% electrophoresis gel as discrete bands at ∼900 bp, with that of XynΔ19 shows a little larger molecular mass than the XynΔ25 (Fig. 1). After the sequence accuracy of the recombinant plasmids had been confirmed, transformed cells containing pET20b-xynΔ19 and pET20b-xynΔ25 were induced to express xylanases. The XynΔ19 and XynΔ25 constructs appeared on the 10% SDS-PAGE gel as discrete bands at ∼34 kDa, with that of XynΔ19 shows a little larger
Discussion
To determine whether the 1–19 (Met1–Ser19) or the 1–25 (Met1–Arg25) residue is the XynB real SP, the XynΔ25 and XynΔ19 were constructed by respectively deleting the first 19 or 25 residues. After expression in E. coli BL21(DE3), the two constructs were assayed and compared for properties. The XynΔ25 had 10 °C higher Topt value, 21.6-times higher thermostability, and 2.3-times higher catalytic efficiency than those of XynΔ19. The XynΔ25 had 302 amino-acid residues (Gln26–Leu302), equivalent to
Acknowledgements
This work was supported by the Natural Science Foundation of China (30972123). The authors are grateful to Ian Riley for his advice on writing the paper and the anonymous reviewers for their sincere suggestions of the paper.
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