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. 2008 Feb 6;3(2):e1551. doi: 10.1371/journal.pone.0001551

Figure 1. Expression and Purification of A Cell Wall Anchored Sialidase.

Figure 1

(A) A vector encoding a cell wall anchored sialidase (accession # gi|50843035) was constructed by inserting a PCR amplified full sialidase gene into the pEcoli-Nterm 6xHN vector (Clontech Laboratories, Inc., Mountain View, CA) at the SaII and HindIII restriction sites. Specific primers including the sense (5′- ATGACTTTGACCACGAAACTGAGCG-3′) and anti-sense primers (5′-TCAGGCAGGGCTCCGGCCCCAGATGC-3′) were designed to clone sialidase from P. acnes (ATCC 6919). The vector, which contains T7/LacO promoter, is derived from the pET system developed by William Studier and colleagues to achieve exceptionally high levels of protein expression in E. coli. Sialidase (arrow) was expressed in E. coli in the absence (lane 1) or presence (lane 2) of (1 mM) IPTG. After IPTG induction, sialidase was successfully expressed in E. coli and shown at about 53.1 kDa on a 10 % SDS-PAGE (arrowhead). (B) Purified sialidase (arrow) was obtained via In-Fusion Ready TALON Express Bacterial Expression and Purification kit (Clontech Laboratories, Inc., Mountain View, CA). (C) The expression and purity of sialidase were confirmed by Nano-LTQ MS/MS mass spectrometry (Thermo Electron Corp. Waltham, MA). A sequenced internal peptide (VVELSDTLMLNSR) of sialidase was presented.