PubMed · 12635255
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1972-07-07. DNA structure. Geometrical versatility.. https://doi.org/10.1038/238015a0
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Microbes produce bioactive secondary metabolites as toxins, pigments, or virulence factors. These specialized compounds are produced by nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), or hybrid NRPS/PKS pathways. The genes encoding NRPS and PKS reside in biosynthetic gene clusters (BGCs), some of which have no identified metabolite associated with them. Characterization of these orphan BGCs could provide insights into potential bioactive compounds that have yet to be discovered. Here, we characterize PA1216, a putative methyltransferase embedded within an NRPS BGC in Pseudomonas aeruginosa strain PAO1. We cloned, expressed, and purified PA1216, and developed an optimized differential scanning fluorimetry assay to measure its thermal stability, demonstrating concentration-dependent stabilization in the presence of established methyltransferase cofactors and inhibitors. We then adapted this assay for high-throughput screening of potential PA1216 substrates, identifying destabilizing compounds, including glycyl-glycine dipeptides, amino esters with aromatic or basic side chains, and N-Boc-protected amino acids. In contrast, sodium salts of organic acids stabilized PA1216. Lastly, we employed AlphaFold to construct a predictive model, revealing that PA1216 contains a Rossmann-like fold and a glycine-rich loop, typical of class I methyltransferases, and we corroborated these secondary structural elements using circular dichroism spectroscopy. Overall, these studies illuminate PA1216 function and establish a platform for characterizing cryptic gene clusters within secondary metabolic pathways.
Resistin, a small cysteine rich protein secreted by adipocytes, has been proposed to be a link between obesity and type II diabetes by modulating the insulin signaling pathway and thus inducing insulin resistance. Resistin protein, with 11 cysteine residues, was not significantly homologous at the amino acid level to any other known cysteine rich proteins. Resistin cDNA derived from human subcutaneous adipose tissue was expressed in Escherichia coli as an N-terminal six-His-tag fusion protein. The overexpressed recombinant resistin was purified to homogeneity from inclusion bodies, after solubilization in 8 M urea, using a metal affinity column. While MALDI-TOF mass spectrometric analysis of the purified protein generated a single peak corresponding to the estimated size of 11.3 kDa, the protein exhibited a concentration-dependent oligomerization which is evident from size exclusion chromatography. The oligomeric structure was SDS-insensitive but beta-mercaptoethanol-sensitive, pointing to the importance of disulfide linkages in resistin oligomerization. Estimation of free cysteine residues using the NBD-Cl assay revealed a concentration- and time-dependent increase in the extent of formation of disulfide linkages. The presence of intermolecular disulfide bond(s), crucial in maintaining the global conformation of resistin, was further evident from fluorescence emission spectra. Circular dichroism spectra revealed that recombinant resistin has a tendency to reversibly convert from alpha-helical to beta-sheet structure as a direct function of protein concentration. Our novel observations on the biophysical and biochemical features of human resistin, particularly those shared with prion proteins, may have a bearing on its likely physiological function.
Today, many valuable proteins can be obtained in sufficient amounts using recombinant DNA techniques. However, frequently the expression of recombinant proteins results in the accumulation of the product in dense amorphous deposits inside the cells, called inclusion bodies. The challenge then is to transform these inactive and misfolded protein aggregates into soluble bioactive forms. Although a number of general guidelines have been proposed, the search for proper reconstitution conditions can be very laborious and time consuming. Here, we suggest a new versatile approach for solubilization and refolding of inclusion body proteins using a water-sodium bis-2-ethylhexyl sulfosuccinate-isooctane reverse micellar system. Instead of amorphous aggregates, a transparent solution is obtained, where refolded protein is entrapped inside the micelles. The entrapped enzyme has native-like secondary structure and catalytic activity. This approach has been implemented with Fusarium galactose oxidase and Stigmatella aurantiaca putative galactose oxidase.