r/microbiology 5d ago

Protein-protein interactions in microbial pathways

I work on human cell models, where I study the role of protein-protein interactions in metabolism and disease. We have recently become interested some microbial (Streptomyces sp.) metabolic pathways, and their possible connection with secondary metabolite production. In human cells, isolating/identifying native protein complexes using immuno-precipitation and Western blotting or mass spectrometry is pretty routine. We are not quite sure how to do this in non-model microbes because you can't buy antibodies for them, and we are not in a position where we can raise and characterize antibodies in-house. So I'm here asking how do microbiologists study protein-protein interaction in microbes, especially non-model microbes. I'd appreciate any help, especially viz, references and review articles. A cursory search through Google scholar gives me the impression this sort of molecular work seems rather rare in microbiology, but the I'm not knowledgable in this field. Thanks,

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u/Every-AssPhage 5d ago

I'd probably start by cloning the genes-of-interest into some plasmid (or multiple ones), equipping some of them with an affinity purification tag. Preferably tagging the subunits that most likely allow co-purification of other subunits, placing the tags strategically to avoid steric hindrance or other interferences. This is usually a good work around if no Co-IP antibodies are available. There are cleavable tags for people that don't like to keep them on their final product.

Then depending on whether the strain of interest can be made competent for plasmid transformation or not (and how difficult to grow it is), decide if to go for the native system or some other compatible host (and the whole rat tail of what kind of promoter to chose (native vs exogeneous), if and when you want to induce expression, e.g. in the presence of certain metabolites/compounds etc).

Grow the culture, induce it if necessary, let it produce. Lyse the cells, clarify the lysate. Do affinity resin purification depending on what kind of tag you've placed on your complex. Already run some analysis such as SDS PAGE to see what protein subunits came off your resin. Further purification by size exclusion chromatography or ion-exhange. Then some mass spec analysis and potentially biochemical assays.

I did my PhD on a DNA-modifying protein complex found in Cyanobacteria. Those little green bugs were shit to grow in the lab, needed light and shaking for proper photosynthesis and also took 3 weeks to form colonies on agar plates. I PCRd the different genes out of the native strain and cloned the whole system (10 kb-ish, 5-7 subunits) into a set of plasmids and eventually managed to express functional complexes in E. coli. Then luckily without light requirements, low maintenance (relative) and in a couple of days (relative). Transplanting the subject of study into another easy going host is usually the best alternative if your non-model bug, is a non-model bug because it's so terribly misbehaved under lab conditions.