Simple and Reliable Method To Precipitate Proteins from Bacterial Culture Supernatant

Abstract

A simple and reliable method for precipitating protein from bacterial culture supernatants based on a pyrogallol red-molybdate-methanol (PRMM) protocol has been developed and applied for the analysis of proteins secreted by a bacterial type III secretion system. PRMM-based precipitation has been shown to be more efficient and robust than are conventional protocols.Recently, bacterial mechanisms for protein secretion have become an important focus of research due to the observation that key virulence factors of bacterial pathogens are secreted into the environment or even translocated into targeted eukaryotic cells. Gram-negative pathogens affecting animals, humans, and plants manipulate targeted host cells by secretion of effector proteins by type III secretion systems (5, 7). Our particular interest is the analysis of protein secretion by Salmonella enterica serovar Typhimurium via the type III secretion system (T3SS) encoded on the Salmonella pathogenicity island 2 (SPI2) (13). Protein secretion via the different T3SSs has been reported to depend on specific environmental factors, and techniques have been established to induce protein secretion into culture supernatants for a variety of T3SSs in vitro (3, 10). However, the standard procedures for protein precipitation of voluminous liquid samples such as culture supernatants harbor several drawbacks, including the handling of large amounts of culture supernatant, toxic chemicals, requirement of high centrifugation forces, low efficiency of precipitation at low protein concentrations, or interfering agents (1, 16). Here, we report on the development of a simple and reliable method for precipitating proteins from bacterial culture supernatant based on a pyrogallol red-molybdate-methanol (PRMM) protocol.Using standard trichloroacetic acid (TCA) and acetonebased protocols for protein precipitation (6, 14), we were unable to reproducibly detect the S. enterica serovar Typhimurium SseB protein, a protein known to be secreted by the S. enterica serovar Typhimurium SPI2-encoded T3SS (2, 11), in culture supernatant (CS) and surface-detached (SD) preparations with satisfactory reliability. In vitro, this particular T3SS is inducible by applying low-pH and magnesium starvation culture conditions (2, 3), but the total amount of Salmonellasecreted proteins under these particular conditions is relatively small. Furthermore, individual protein concentrations within this fraction can vary substantially (11).To optimize the protein yield in precipitates, a recently described method for protein precipitation from chromatographic samples (1, 8) was adapted to our experimental setup. For induction of protein secretion by the SPI2-encoded S. enterica serovar Typhimurium T3SS, the strains to be analyzed were grown as described previously (11) to an optical density at 600 nm of approximately 1.2. To obtain the CS fraction, the cultures were centrifuged for 15 min at 5,000 ϫ g, and the supernatant of each sample was collected and filtered though a 0.22-m-pore-size filter to remove re...

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