Significant Enhanced Expression and Solubility of Human Proteins in Escherichia coli by Fusion with Protein S from Myxococcus xanthus

Abstract

Protein S is a major spore coat protein of Myxococcus xanthus, consisting of two homologous domains, the N-terminal domain (NTD) and the C-terminal domain, both of which contain a Ca 2؉ -binding site. Protein S tightly binds to myxospores in a Ca 2؉ -dependent manner. Here, we constructed a novel expression vector, pCold-PST, encoding two tandem repeat NTDs (PrS 2 ). By using this vector, a number of human proteins that were expressed at low levels or in insoluble forms by a pET vector were expressed not only at high levels but also in soluble forms. We also demonstrated that an Escherichia coli protein tagged with PrS 2 fully retained its function, indicating that it is folded independently from the tag. This technology not only allows simple, one-step protein purification using myxospores, but can also be used for the identification of proteins interacting with a protein of interest and will prove immensely useful for structural studies of proteins which are difficult to produce or are insoluble.

Noncognate Mycobacterium tuberculosis Toxin-Antitoxins Can Physically and Functionally Interact

“…pET21c-MazF-mt1, pET21c-MazF-mt3, pET21c-VapCmt24, and pET21c-VapC-mt25 were each digested with NdeI and BamHI, and the toxin-containing DNA fragment was ligated into the corresponding sites in the pCold-PST vector (25) to create the respective protein S-tagged toxins. Plasmid pCold-TF-MazE-mt1 was constructed after digestion of pET28a-MazE-mt1 with NdeI and BamHI and ligated into the corresponding sites in the pCold-TF vector (Takara-Bio, Inc.) to express MazE-mt1 fused to trigger factor (TF), a ribosomeassociated chaperone protein that facilitates co-translational folding of nascent polypeptides.…”

“…Purification of Protein S-tagged Toxins-PST-MazF-mt1, PST-MazF-mt3, PST-VapC-mt24, and PST-VapC-mt25 proteins tagged at the N terminus were purified from the BL21(DE3) strain carrying pCold-PST-MazF-mt1, pCold-PST-MazF-mt3, pCold-PST-VapC-mt24, or pCold-PSTVapC-mt25, respectively, using myxospores as described previously (25). Briefly, expression of the protein S-tagged proteins was induced at an A 600 of 0.6 by adding IPTG to a final concentration of 1 mM and continuing to grow the cells at 15°C for 16 h. The cells were then harvested, lysed by sonication, and incubated with myxospores in 1 mM CaCl 2 , 50 mM Tris-HCl (pH 8.0), 50 mM KCl, 5% glycerol at 4°C for 1 h to facilitate binding of the protein S-tagged proteins to the myxospores.…”

High yield expression of proteins in <i>E. coli</i> for NMR studies

“…Thus obtained crude cell lysate is amenable for NMR studies making this a rapid high yield protein production method [16]. Enhanced protein expression is observed by cold shock induction in E. coli transformed with pCold-PST vectors in a similar fashion as described above with PrS 2 tags having affinity to myxospores [18].…”

“…Multiple tags can be added together in different combination for a particular protein to get better result on these issues [7]. Various studies report high yield target protein expression with Maltose binding protein (MBP) [19], Glutathione-S transferase (GST) [20], Thioredoxin fusion (TRX) [21], Hexahistidine His6-Tag [19,22], Small ubiquitinmodifier fusion (SUMO) [21], N-utilizing substance A (NusA), Protein S tag (PrS2) [18], Solubility-enhancing tag (SET) [23], Disulfide bond C (DsbC), Seventeen kilodalton protein (Skp), Phage T7 protein kinase (T7PK), Protein G B1 domain (GB1), Protein A IgG ZZ repeat domain (ZZ) [24], histidine tagged Ubiquitin (Ub) fusion and histidine tagged deubiquitylating enzyme (DUB) fusion [25]. Recent studies propose the Outer membrane protein A (OmpA), Outer membrane protein F (OmpF) and Osmotically inducible protein Y (OsmY) as fusion partners secreting protein into the growth medium of E. coli depending on culture conditions via periplasm secretion.…”

Segmental isotope labeling of proteins for NMR structural study using a protein S tag for higher expression and solubility

“…Previously, we have shown that when human proteins are tagged with tandem repeated N-terminal domains of protein S (PrS 2 ), their expression and solubility are significantly enhanced (Kobayashi et al 2009). Moreover, PrS 2 -tagged E. coli OmpR was found to fully retain its function as a transcription regulator, indicating that the PrS 2 -tag does not interfere with the structure and function of the target protein that is fused to the tag (Harlocker et al 1995; Kobayashi et al 2009).…”