title: Rapid in vitro protein synthesis pipeline: a promising tool for cost-effective protein array design.
abstract: Several protein expression systems for construction of protein arrays have been established in recent years. However, current protocols for protein synthesis are still time consuming, laborious and expensive. This study has established an alternative workflow that covers rapid construction of expression cassettes, in-tube and on-membrane synthesis of recombinant proteins, and straightforward screening of synthesized proteins. Eighteen membrane associated eukaryotic proteins and two secretory complement regulators (C1 inhibitor and vitronectin) were included in the study. To generate hybrid genes, double-overlap extension PCR was employed to fuse the 5' fragment (consisting of a T7 promoter and a species independent translation sequence), ORFs of the target proteins, and the 3' fragment (encompassing GFP fusion, Myc-tag and stop codon). OE-PCR generated fragments were directly mixed with the Leishmania torentolae lysate (translation mix) for protein synthesis. In order to establish a cheap and user-friendly alternative to existing cell-free protein array techniques, PCR products were spotted on the hydrophobic substrate (PVDF membrane), air-dried and covered with only 2 μL of translation mix. All synthesized proteins were spontaneously immobilized on the membrane due to the hydrophobic interaction between C-terminally fused GFP and PVDF. Synthesis and immobilization of proteins were confirmed simply by assessing the GFP chromophore under a laser scanner or a fluorescent microscope.
authors.affiliation: Laboratory of Biomedical Microbiology and Immunology, Department of Microbiology and Immunology, University of Veterinary Medicine and Pharmacy, Komenskeho 73, 04181, Kosice, Slovakia. mangeshbhide@me.com.
authors.forename: Mangesh
authors.initials: M
authors.lastname: Bhide
authors.forename: Satheesh
authors.initials: S
authors.lastname: Natarajan
authors.forename: Stanislav
authors.initials: S
authors.lastname: Hresko
authors.forename: Carmen
authors.initials: C
authors.lastname: Aguilar
authors.forename: Elena
authors.initials: E
authors.lastname: Bencurova
mesh_terms: D017931:DNA Primers; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D066298:In Vitro Techniques; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D016133:Polymerase Chain Reaction; D040081:Protein Array Analysis; D015202:Protein Engineering / Q000191:economics* / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins