title: Enhancing the thermal stability of a single-chain Fv fragment by in vivo global fluorination of the proline residues.
abstract: Single-chain Fv (scFv) format protein is a commonly used analytical tool for diagnostic and therapeutic applications. The usage of scFv antibody fragments in therapeutic applications has demonstrated that they need to have high thermostability. Many rational or irrational methods have been described erstwhile to engineer or improve the stability of scFv proteins by modifications of natural amino acid. Here we have demonstrated an alternate strategy to efficiently improve the thermostability of scFvs by non-canonical amino acid. Previously, fluoroprolines have been proven as a choice to tune the stability of many polypeptides and few globular proteins. Hence we exploited the usage of fluoroproline to enhance the thermal stability of scFv by replacing the natural proline on the framework regions of scFv that influence the folding or stability. To demonstrate our approach, a bacterial cytoplasmic foldable and humanized anti-c-Met scFv (hu-MscFv) was used. The hu-MscFv proline sites were successfully incorporated with (2S,4R)-4-fluoroproline without affecting its structure and function by the in vivo residue specific global replacement method which exploits bacterial auxotrophic system. The time-dependent temperature effect on the activity of fluorinated hu-MscFv revealed its increased stability at 40 °C along with improved half-life than the hu-MscFv with natural proline. Further model based structure analysis on hu-MscFv with fluoroproline indicated that the fluorine atoms were able to establish new favourable dipole interactions with neighbouring polar groups in their local micro environments that rationalizes its improved thermostability. Moreover the scFv sequence based statistical analysis strongly supports the fact that this method can be applied to any target scFv, since they contain high frequency conserved proline sites in their framework regions.
authors.affiliation: Dept of Chemical Engineering, Pusan National University, Busan, Republic of Korea.
authors.forename: Selvakumar
authors.initials: S
authors.lastname: Edwardraja
authors.forename: Sokalingam
authors.initials: S
authors.lastname: Sriram
authors.forename: Raghunathan
authors.initials: R
authors.lastname: Govindan
authors.forename: Nediljko
authors.initials: N
authors.lastname: Budisa
authors.forename: Sun-Gu
authors.initials: SG
authors.lastname: Lee
mesh_terms: D002853:Chromatography, Liquid; D013058:Mass Spectrometry; D008958:Models, Molecular; D011392:Proline / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D055550:Protein Stability; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D057127:Single-Chain Antibodies; C431803:fluoro-proline; D011392:Proline