title: Expression, purification and antimicrobial activity of puroindoline A protein and its mutants.
abstract: Wheat puroindoline proteins, PINA and PINB, play key roles in determining wheat grain hardness as well as in defending the plant against pathogens. PINA has much greater membrane-binding property and antimicrobial activity because it contains more tryptophan residues in the unique tryptophan-rich domain (TRD). In order to obtain proteins with higher antimicrobial activity, mutants of PINA containing two or three copies of TRD, designated ABBC and ABBBC, respectively, were constructed and expressed in E. coli Rosetta-gami (DE3). Metal affinity chromatography was used to purify the soluble affinity-tagged recombinant proteins. The secondary structures of the recombinant proteins were predicted by the online program Protein Homology/analog Y Recognition Engine v2.0 and experimentally assessed using circular dichroism. Minimum inhibition concentration tests and fluorescence microscope analyses were employed to evaluate the antimicrobial activities of the mutants. The results showed that the purified recombinant ABBC was correctly folded and presented significantly higher antimicrobial activities against E. coli and S. aureus than wild-type PINA, suggesting its potential use as an antimicrobial agent. The results also confirmed that TRD is a determinant of the antimicrobial activity of PINA and demonstrated that it is feasible to enhance the antimicrobial activity of PINA by adding one copy of TRD.
authors.affiliation: China-UK HUST-RRes Genetic Engineering and Genomics Joint Laboratory, Genetic Engineering International Cooperation Base of MoST of China, Key Laboratory of Molecular Biophysics MoE of China, Huazhong University of Science and Technology, Wuhan, 430074, People's Republic of China.
authors.forename: Yingjie
authors.initials: Y
authors.lastname: Miao
authors.forename: Ling
authors.initials: L
authors.lastname: Chen
authors.forename: Cheng
authors.initials: C
authors.lastname: Wang
authors.forename: Yajuan
authors.initials: Y
authors.lastname: Wang
authors.forename: Qian
authors.initials: Q
authors.lastname: Zheng
authors.forename: Chunbao
authors.initials: C
authors.lastname: Gao
authors.forename: Guangxiao
authors.initials: G
authors.lastname: Yang
authors.forename: Guangyuan
authors.initials: G
authors.lastname: He
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000494:pharmacology; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D015870:Gene Expression; D005818:Genetic Engineering; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000254:growth & development; D013329:Structure-Activity Relationship; D014908:Triticum / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C082942:puroindoline protein, Triticum aestivum; D014364:Tryptophan