SDS-PAGE

[Mycozoo]

Hello,

I'm trying to find a method to extract enzymes from filamentous fungi. I'm not a molecular biologist by any means so I have no idea which methods does what.

I came across SDS-PAGE, which as I understand separates enzymes based on weight?

To give some context, some of these fungi don't have annotated genomes. So basically what I'm asking is SDS-PAGE a good method to identify which enzymes exist in a fungal sample?

[CharonY]

SDS-PAGE only separates, but alone does not identify what proteins you separated. You need to combine with a targeted approach such as Western blot, but that requires a suitable antibody and knowledge what you are looking for. Mass spectrometry is also theoretically possible, but without genome data you will have to rely on closely related species or need to conduct de novo sequencing which is not that great most of the time.

[Mycozoo]

@CharonY Thanks for the response! Could you combine it with LC-MS/MS? Or perhaps use LC-MS/MS alone to identify the enzymes?

[CharonY]

You can use mass spectrometry, but as I mentioned before, without genomic information it will be difficult to identify all but the most conserved proteins. De novo sequencing is a possibility in which you use MS to try to  deduce the AA sequence. But that is often tricky and often does not work well straight from complex mixtures. One could try to run a 2D-gel and try to isolate enough of a particular protein of interest but it is not really something that would work well in high-throughput.

[Mycozoo]

Okay. What about using a reference genome of a closely related species to create a probe looking for similar enzymes? Is that getting more into qPCR?
Sorry for these ongoing questions, I do appreciate your answers though!

[Dagl1]

8 minutes ago, Mycozoo said:

Okay. What about using a reference genome of a closely related species to create a probe looking for similar enzymes? Is that getting more into qPCR?
Sorry for these ongoing questions, I do appreciate your answers though!

qPCR is involved in RNA quantification. It is not used to extract enzymes.

@CharonY How unreliable is sequencing a new species? And how does it become more reliable, just by more people confirming the same sequence? I thought that with good enough sequencing you would be able to identify at least coding regions (of course, repeats probably remain a problem), am I overestimating sequencing ability (I suppose it could also just be how a matter of how financially (un)viable adequate sequencing is in this case)?

[CharonY]

5 minutes ago, Dagl1 said:

qPCR is involved in RNA quantification. It is not used to extract enzymes.

@CharonY How unreliable is sequencing a new species? And how does it become more reliable, just by more people confirming the same sequence? I thought that with good enough sequencing you would be able to identify at least coding regions (of course, repeats probably remain a problem), am I overestimating sequencing ability (I suppose it could also just be how a matter of how financially (un)viable adequate sequencing is in this case)?

I think you misunderstood my post (which had a typo, I meant de novo sequencing, not de novo synthesis). De novo sequencing in protein sciences refers to sequencing an isolated protein via fragmentation in the MS. From there, at least in principle, you can deduce the amino acid sequence and use that sequences for further analyses. It does not refer to  DNA sequencing.

 

[Dagl1]

52 minutes ago, CharonY said:

I think you misunderstood my post (which had a typo, I meant de novo sequencing, not de novo synthesis). De novo sequencing in protein sciences refers to sequencing an isolated protein via fragmentation in the MS. From there, at least in principle, you can deduce the amino acid sequence and use that sequences for further analyses. It does not refer to  DNA sequencing.

 

Ahh I see, thanks. 
However I do wonder, wouldn't first sequencing the genetic code give you predicted proteins (of course not perfect if there's splicing, but I have no idea about fungi). Then it should be easier to identify the proteins in MS (as you said that this can not too great)?

[CharonY]

6 minutes ago, Dagl1 said:

Ahh I see, thanks. 
However I do wonder, wouldn't first sequencing the genetic code give you predicted proteins (of course not perfect if there's splicing, but I have no idea about fungi). Then it should be easier to identify the proteins in MS (as you said that this can not too great)?

Sequencing using next-gen method is relatively affordable, but assembly can be tricky. And without a related genome as scaffold it is unlikely to close. You can still search for ORFs in an incomplete genome, but at some point you would have to ask yourself whether it would be worth the money and effort. I.e. what is the actual research question? If the goal is to identify a protein with a specific function it seems like a rather useless endeavour to sequence the genome.

[hypervalent_iodine]

If the OP has specific enzymes in mind, and a closely related fungi with an annotated genome exists, they could use that to design primers and sequence the enzymes. It’s relatively easy (I can do it for instance, and I only pretend to sometimes be a biologist) and cheap, though it does rely pretty heavily on good primer design and some knowledge of codon bias. NGS seems like overkill for something like this, for the reasons CharonY mentioned. 

[CharonY]

7 hours ago, hypervalent_iodine said:

If the OP has specific enzymes in mind, and a closely related fungi with an annotated genome exists, they could use that to design primers and sequence the enzymes. It’s relatively easy (I can do it for instance, and I only pretend to sometimes be a biologist) and cheap, though it does rely pretty heavily on good primer design and some knowledge of codon bias. NGS seems like overkill for something like this, for the reasons CharonY mentioned. 

It is really unclear what OP wants. If the goal is e.g. to identify enzymes with certain activity the standard procedure would be to test protein fractions for said activity followed by deeper characterization of these fractions, for example. If testing for absence or presence of certain enzymes DNA-based studies would generally make more sense.

[Mycozoo]

On 2/14/2020 at 6:38 AM, hypervalent_iodine said:

If the OP has specific enzymes in mind, and a closely related fungi with an annotated genome exists, they could use that to design primers and sequence the enzymes. It’s relatively easy (I can do it for instance, and I only pretend to sometimes be a biologist) and cheap, though it does rely pretty heavily on good primer design and some knowledge of codon bias. NGS seems like overkill for something like this, for the reasons CharonY mentioned. 

On 2/14/2020 at 1:58 PM, CharonY said:

It is really unclear what OP wants. If the goal is e.g. to identify enzymes with certain activity the standard procedure would be to test protein fractions for said activity followed by deeper characterization of these fractions, for example. If testing for absence or presence of certain enzymes DNA-based studies would generally make more sense.

To get at both of your questions, the goal is to identify keratinases and learn which ones are active during keratin degradation. 

 

[CharonY]

Novel ones or similar to known ones?

[Mycozoo]

I imagine similar to known ones? As I understand it (and perhaps inaccurately), keratinases are different in just about every fungus that has them. Because there has been a lot of work looking at keratin degradation in Aspergillus fumigatus, I figured I could potentially use it as a reference even though the fungi of interest in are reptile pathogens. 

[CharonY]

I do not know how conserved they are, but if they are not, PCR based amplification/identification may not work out. What is often done to find enzymes with desired functions is to isolate protein fractions, test them for desired activity and then sub-fractionate those until you get a decently pure sample to analyze further.

[BabcockHall]

SDS PAGE denatures proteins, and not every protein that is denatured can be renatured.  If you need active enzymes and you want to separate by molecular weight, then gel filtration might be an appropriate technique.  However, it is often the case that obtaining a pure protein requires more than one method of separation.