title: Cloning, expression and purification of penicillin-binding protein 3 from Pseudomonas aeruginosa CMCC 10104.
abstract: Penicillin-binding protein 3 (PBP3) of Pseudomonas aeruginosa is the primary target of β-lactams used to treat pseudomonas infections. Meanwhile, structure change and overproduction of PBP3 play important roles in the drug resistance of P. aeruginosa. Therefore, studies on the gene and structure of PBP3 are urgently needed. P. aeruginosa CMCC 10104 is a type culture strain common used in China. However, there is no report on its genomic and proteomic profiles. In this study, based on ftsI of P. aeruginosa PAO1, the gene encoding PBP3 was cloned from CMCC 10104. A truncated version of the ftsI gene, omitting the bases encoding the hydrophobic leader peptide (amino acids 1-34), was amplified by PCR. The cloned DNA shared 99.76% identity with ftsI from PAO1. Only four bases were different (66 C-A, 1020 T-C, 1233 T-C, and 1527 T-C). However, there were no differences between their deduced amino acid sequences. The recombinant PBP3 (rPBP3), containing a 6-histidine tag, was expressed in Escherichia coli BL21 (DE3). Immobilized metal affinity chromatography (IMAC) with Ni(2+)-NTA agarose was used for its purification. The purified rPBP3 was identified by SDS-PAGE and western blot analysis, and showed a single band at about 60kDa with purity higher than 95%. The penicillin-binding assay indicated that the obtained rPBP3 was functional and not hindered by the presence of the C-terminal His-tag. The protocol described in this study offers a method for obtaining purified recombinant PBP3 from P. aeruginosa CMCC 10104.
authors.affiliation: College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310018, China.
authors.forename: Yan Dong
authors.initials: YD
authors.lastname: An
authors.affiliation: Zhejiang A&F University, Hangzhou 311300, China.
authors.forename: Qi Zhen
authors.initials: QZ
authors.lastname: Du
authors.affiliation: College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310018, China.
authors.forename: Li Yan
authors.initials: LY
authors.lastname: Tong
authors.affiliation: College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310018, China.
authors.forename: Zhao Wu
authors.initials: ZW
authors.lastname: Yu
authors.affiliation: College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310018, China. Electronic address: gongxingwen@163.com.
authors.forename: Xing Wen
authors.initials: XW
authors.lastname: Gong
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D046915:Penicillin-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D010406:Penicillins / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D021382:Protein Sorting Signals / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D017384:Sequence Deletion; D018440:beta-Lactam Resistance / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins
keywords: Cloning; Expression; Penicillin-binding protein 3; Pseudomonas aeruginosa; Purification