An evaluation of in vitro protein–protein interaction techniques: Assessing contaminating background proteins

Abstract

Determination of protein-protein interactions is an important component in assigning function and discerning the biological relevance of proteins within a broader cellular context. In vitro protein-protein interaction methodologies, including affinity chromatography, coimmunoprecipitation, and newer approaches such as protein chip arrays, hold much promise in the detection of protein interactions, particularly in well-characterized organisms with sequenced genomes. However, each of these approaches attracts certain background proteins that can thwart detection and identification of true interactors. In addition, recombinant proteins expressed in Escherichia coli are also extensively used to assess protein-protein interactions, and background proteins in these isolates can thus contaminate interaction studies. Rigorous validation of a true interaction thus requires not only that an interaction be found by alternate techniques, but more importantly that researchers be aware of and control for matrix/support dependence. Here, we evaluate these methods for proteins interacting with DmsD (an E. coli redox enzyme maturation protein chaperone), in vitro, using E. coli subcellular fractions as prey sources. We compare and contrast the various in vitro interaction methods to identify some of the background proteins and protein profiles that are inherent to each of the methods in an E. coli system.

Engineering Escherichia coli BL21(DE3) Derivative Strains To Minimize E. coli Protein Contamination after Purification by Immobilized Metal Affinity Chromatography

“…Mass spectrometry (MS) analysis of the target protein elution fractions revealed that the following E. coli host proteins also coeluted in significant amounts: DnaK, GlmS, AceE, EF-Tu, ArnA, RnaseE, AtpF, the Rho transcription terminator, CRP, and SlyD (data not shown). Several independent studies, performed using different conditions or different E. coli strains, also reported many of the same contaminants after Ni ϩ2 affinity chromatography (5,13,18). The most common E. coli proteins listed in these previous reports were CRP, Fur, ArgE, DnaK, SlyD, GlmS, GlgA, ODO1, ODO2, Can (YadF), ArnA (YfbG), AceE, GroES, and GroEL.…”

“…Protein samples of approximately 1 mg were added to 20 l of trypsin reaction buffer (50 mM Tris-HCl, 20 mM CaCl 2 , pH 8) and digested overnight at 37°C with trypsin (New England BioLabs) at a protein-to-protease ratio of 20:1. Online liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) analyses of digested fractions using an Agilent 6330 Ion Trap mass spectrometer with an integrated C 18 chromatin immunoprecipitation-nanoelectrospray ionization (C 18 ChIP/nano-ESI) interface were performed as described previously (40). Protein separation, digestion, and peptide analysis were repeated in triplicate for each sample.…”

Temporal Profile of Cerebrospinal Fluid, Plasma, and Brain Interleukin-6 After Normothermic Fluid-Percussion Brain Injury: Effect of Secondary Hypoxia

“…In regard to the present findings with secondary hypoxia, Bramlett et al (1999b) reported that this complicated TBI model leads to a larger cortical contusion and aggravated hippocampal damage associated with a more robust inflammatory response. In previous studies, structural damage and an aggravated inflammatory response has been correlated with elevated IL-6 levels after brain injury (Howell et al, 2006;Yan et al, 2012). After stroke, for example, elevated levels of IL-6 are associated with the infarct size and poor outcome (Vila et al, 2000;Smith et al, 2004;Waje-Andreassen et al, 2005).…”

“…After stroke, for example, elevated levels of IL-6 are associated with the infarct size and poor outcome (Vila et al, 2000;Smith et al, 2004;Waje-Andreassen et al, 2005). In this regard, IL-6 levels are considered a highly specific biomarker of TBI severity and long-term outcome (van Griensven et al, 2003;Howell et al, 2006). In addition, IL-6 plays an important role in activating and recruiting immune cells and has been reported to suppress TNF-a and IL-1-b production and stimulate nerve growth factor (NGF) production (Kushima et al, 1992;Juttler al., 2002).…”

Identification of Novel SHPS-1-associated Proteins and Their Roles in Regulation of Insulin-like Growth Factor-dependent Responses in Vascular Smooth Muscle Cells

“…S6). Although a number of novel SHPS-1/CD binding partners were identified in the present study using the mRNA display and TAP tag method and the interactions were confirmed with co-immunoprecipitation, previous studies have shown that the evaluation of in vitro protein-protein interactions by multiple orthogonal techniques is essential (49). Thus, these interactions need to be validated using other orthogonal technique.…”