Activity

Proteins naturally expressed in eukaryotic organisms often require host chaperones, binding partners, and posttranslational modifications for correct folding. Ideally the heterologous expression system chosen should be as similar to the natural host as possible. For example, mammalian proteins should be expressed in mammalian expression systems. Reversan However, this does not guarantee a protein will be expressed in a sufficient high yield for structural or biochemical studies or antibody generation. Often a screening process is undertaken in which many parameters including truncations, point mutations, investigation of orthologs, fusion to peptide or protein tags at the N- or C-terminus, the coexpression of binding partners, and even culture conditions are varied to identify the optimal expression conditions. This requires multiparallel expression screening in mammalian cells similar to that already described for E. coli expression. Here we describe in detail a multiparallel method to express proteins in mammalian suspension cells by transient transfection in 24-well or 96-well blocks.This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the BacMam system. This eukaryotic expression system was selected and a screening process established in 2016 to enable production of highly challenging human integral membrane proteins (IMPs), which are a significant component of our target list. Here, we discuss our recently developed platform for identifying expression and monodispersity of IMPs from 3 mL of HEK293 cells.This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the baculovirus expression vector system (BEVS). This eukaryotic expression system was selected and a screening process established in 2007 as a measure to tackle the more challenging kinase, RNA-DNA processing, and integral membrane protein families on our target list. Here, we discuss our platform for identifying soluble proteins from 3 mL of insect cell culture and describe the procedures involved in producing protein from liter-scale cultures.In Chapter 3 , we described the Structural Genomics Consortium (SGC) process for generating multiple constructs of truncated versions of each protein using LIC. In this chapter we provide a step-by-step procedure of our E. coli system for test expressing intracellular (soluble) proteins in a 96-well format that enables us to identify which proteins or truncated versions are expressed in a soluble and stable form suitable for structural studies. In addition, we detail the process for scaling up cultures for large-scale protein purification. This level of production is required to obtain sufficient quantities (i.e., milligram amounts) of protein for further characterization and/or structural studies (e.g., crystallization or cryo-EM experiments). Our standard process is purification by immobilized metal affinity chromatography (IMAC) using nickel resin followed by size exclusion chromatography (SEC), with additional procedures arising from the complexity of the protein itself.Structural genomics groups have identified the need to generate multiple truncated versions of each target to improve their success in producing a well-expressed, soluble, and stable protein and one that crystallizes and diffracts to a sufficient resolution for structural determination. At the Structural Genomics Consortium, we opted for the ligation-independent cloning (LIC) method which provides the throughput we desire to produce and screen many proteins in a parallel process. Here, we describe our LIC protocol for generating constructs in 96-well format and provide a choice of vectors suitable for expressing proteins in both E. coli and the baculovirus expression vector system (BEVS).With a growing amount of structural information of proteins, deciphering the linkage between the structure and function of these proteins is the next important task in structural genomics. To characterize the function of an enzyme at molecular level, placing a reporter on the active site of an enzyme can be a strategy to examine the dynamics of the interaction between enzyme and its substrate/inhibitor. In this chapter, we describe an approach of active-site labeling of enzyme for this purpose. Provided with the fabrication of a fluorescein-labeled AmpC β-lactamase as an example, we herein depict the methodology of a structure-based selection of the location in an enzyme’s active site for bioconjugation and the preparation of the active-site labeled enzyme.Exploring how combinatorial mutations can be combined to optimize protein functions is important to guide protein engineering. Given the vast combinatorial space of changing multiple amino acids, identifying the top-performing variants from a large number of mutants might not be possible without a high-throughput gene assembly and screening strategy. Here we describe the CombiSEAL platform, a strategy that allows for modularization of any protein sequence into multiple segments for mutagenesis and barcoding, and seamless single-pot ligations of different segments to generate a library of combination mutants linked with concatenated barcodes at one end. By reading the barcodes using next-generation sequencing, activities of each protein variant during the protein selection process can be easily tracked in a high-throughput manner. CombiSEAL not only allows the identification of better protein variants but also enables the systematic analyses to distinguish the beneficial, deleterious, and neutral effects of combining different mutations on protein functions.Skin fibrosis is one of the major features of scleroderma. WNT/β-catenin signaling is associated with the progression of skin fibrosis. In this study, we aimed to determine the effect of icaritin (IT), a natural compound, on scleroderma-related skin fibrosis and its mechanisms. We found that IT could reduce the expression of COL1A1, COL1A2, COL3A1, CTGF, and α-SMA in human foreskin fibroblasts (HFF-1 cells), scleroderma skin fibroblasts (SSF cells), and TGF-β-induced HFF-1 cells. Wnt/β-catenin signaling was shown to be suppressed by IT. Additionally, IT activated AMPK signaling in HFF-1 cells. In conclusion, IT has an anti-skin fibrotic effect through activation of AMPK signaling and inhibition of WNT/β-catenin signaling. Our findings indicate the potential role of IT in the treatment of scleroderma and provide novel insight for the selection of drug therapy for scleroderma.