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Title: Novel human cell expression method reveals the role and prevalence of posttranslational modification in non-muscle tropomyosins
Authors: Carman, Peter J.
Keywords: tế bào người; biến đổi; hậu dịch mã; tropomyosin
Issue Date: 2021
Publisher: bioRxiv
Abstract: Biochemical studies require large protein quantities, which are typically obtained using bacterial expression. However, the folding machinery of bacteria is inadequate for many mammalian proteins, which additionally undergo posttranslational modifications (PTMs) that bacteria, yeast, or insect cells cannot perform. Many proteins also require native N- and C-termini and cannot tolerate extra tag amino acids for function. Tropomyosin, a coiled coil that decorates most actin filaments in cells, requires both native N- and C-termini and PTMs, specifically N-terminal acetylation, to polymerize along actin filaments. Here, we describe a new method that combines native protein expression in human cells with an intein-based purification tag that can be precisely removed after purification. Using this method, we expressed several non-muscle tropomyosin isoforms. Mammalian cell-expressed tropomyosins are functionally different from their E. coli-expressed counterparts, display multiple types of PTMs, and can form heterodimers. This method can be extended to other proteins, as demonstrated here for a synuclein.
URI: http://dlib.hust.edu.vn/handle/HUST/23851
Link item primary: https://www.biorxiv.org/content/10.1101/2021.04.05.438513v2.full.pdf+html
Appears in Collections:OER - Kỹ thuật hóa học; Công nghệ sinh học - Thực phẩm; Công nghệ môi trường
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