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> But when they instructed microorganisms to make their creations in the labs, none of the 150 designs worked. “They didn’t fold at all: they were just gunk at
by adriand 4y ago
> But when they instructed microorganisms to make their creations in the labs, none of the 150 designs worked. “They didn’t fold at all: they were just gunk at the bottom of the test tube,” says Baker.
When it works properly (when they fold), what do you see in the test tube instead? Does it no longer look like “gunk”? What I mean is, is this difference visible to the naked eye?
- flobosg 4y agoWhen you express recombinant proteins in bacteria, you grow them and then let them produce your protein of interest. Then you destroy the bacteria and separate the soluble parts (where you hope your protein stays) from the insoluble ones (protein aggregates, cell membrane components, etc.), usually by ultracentrifugation. The soluble portion (or supernatant) is a clear liquid, while the insoluble fraction (or precipitate) is a whiteish, gunky solid. So, to answer your question, soluble proteins would look clear. However, to actually check for the presence of your protein of interest in each fraction you load them in a polyacrylamide gel and pass current through it to separate all proteins in the mixture, using a method called electrophoresis[1]. [1]: https://en.wikipedia.org/wiki/Polyacrylamide_gel_electrophoresis https://en.wikipedia.org/wiki/Polyacrylamide_gel_electrophor.... In the first picture of the article each band represents one (or several, if you’re unlucky!) protein of a determined molecular weight or size.
- radicalOH 4y agoIs SDS-PAGE the normal way or do they actually do a western or ELISA?
- throwawaymaths 4y agoThe first step of a western is SDS-PAGE.
- DrAwdeOccarim 4y agoTo follow on, typically proteins produced this way have a tag, or handle, to specifically pull it out from all the other proteins. A typica tag is 6 x Histidine amino acids at the beginning of the protein. These histidines bind to nickel, which is immobilized on a resin. You can use antibodies against the 6xHis to Western for your protein of desire without needing a protein specific antibody.
- flobosg 4y agoSDS-PAGE is the normal way, at least for an initial test expression. For a Western blot or ELISA you need antibodies specific to your protein, which you probably don’t have. An easier way to identify, for example, would be to cut the SDS-PAGE band and perform mass spectrometry on it to try to elucidate its protein sequence.
- dekhn 4y agoAs an aside, in the first real purification and isolation of a protein (Rho), there is a figure of a test tube with what just looks like liquid, and a caption "Rho protein in solution". I imagine some soluble proteins actually make the solution look colorful (autofluorescence) but at the concentrations required you'd be pushing up against aggregation
- flobosg 4y agoCofactor- or chromophore-binding proteins are usually colored, which is quite useful during expression and purification. The quintessential example is GFP or GFP-tagged proteins.
- COGlory 4y agoNothing. If they fold well and are water soluble, the solution remains transparent because the protein remains solvated. If they don't fold well, they expose hydrophobic regions that then bind to the hydrophobic regions on neighboring proteins, causing a chain reaction that results in a solid chunk of protein that isn't dissolved. Not all proteins are water soluble in nature, however. Plenty of membrane proteins and other proteins exist that don't fold well when expressed in a micro-organism.