Recently I have come across a protein expressed&secreted by insect cells. The protein was heterologous exhibiting a broad/smeared band in SDS-PAGE gel. I have tried glycoside hydrolase to remove the glycosylation, but without success: the overall molecular weight has reduced while the heterogeneity remained similar. Has anyone met the same problem before and solved it in the end?
Thanks a lot.
Not yet. Thank you very much for this information. Do you have suggested conc. and time of addition? Or probably some reference that I can resort to? Thanks again for this precious suggestion!
Hi Yutao,
Which glycoside hydrolase did you use and in which conditions did you perform tour deglycosylation reaction? Usually, PNGaseF in denaturing conditions should result in complete removal of N-glycosylation. Keep in mind that other post-translational modifications can also result in smearing of the protein, e.g. O-glycans. These are not removed by e.g. PNGaseF or EndoH or EndoF enzymes.
Glycosylated proteins rarely look homogenous on SDS-PAGE, especially from insect cells. What is the end-use of your protein and is it really necessary to reduce this heterogeneity?
If you do need to reduce the heterogeneity you can either block some glycosylation pathways with e.g. kifunesine in your media or strip away different levels of the glycosylation enzymatically from your purified proteins with e.g. PNGase or EndoH (should be particularly effective on high-mannose glycoslylation).
Blocking/removing glycosylation from your proteins may reduce your expression/secretion levels or the solubility of your stripped protein so it can be better in some cases to retain some level of glycosylation.
Thank you for your suggestions, Steven and Nick! The secretion-expressed protein in insect cells was meant for crystallization and therefore it is pivotal to keep the sample homogeneity. I've tried PNGase and cocktails as suggested by the manufacturer's intructions, but the result was not satisfactory. I'll try the inhibition of glycosylation as suggested above and discuss with you afterwards. Good luck~ Thanks again for your suggestions!
Hi Yutao, there are a lot of publications that have protocols for treating insect cell cultures with tunicamycin. Below is the earliest one I could find.
But bear in mind that tunicamycin only inhibits N-linked glycosylation and can lead to lower yields of recombinant proteins.
http://mcb.asm.org/content/3/12/2156.full.pdf
Hi Yutao,
Tunicamycin block glycosylation before the glycan is attached to the protein. It may lead to missfolded protein. How many N-glycosylation sites does your protein have? If you add kifunesine you block processes in the golgi where trimming and modifications take place this can help when you deglycosylate your protein. You need to deglycosylate the native protein which is less efficient. Kifunesine will make the deglycosylation more efficient and you can use EndoH or EndoF1. Deglycosylataion with PNGaseF change the Asn to an Asp whilst EndoH and F1 leave a GlcNac at the glycosylated site.
Thank you all, for your suggestions! I'll take trial experiments and hopefully there will be good result.
p.s. I wonder if there would be O-linked glycosylation in normal insect-expression system? Or is the O-linked glycosylation only existing in mammanian expression systems? on the other hand, If I change the secretion-expression method to intracellular-expression method by truncating the secretion-signal sequence in protein, could I avoid the glycosylation modification to my protein?
N-glycosylation is important for the protein to fold correctly. If you don"t want any glycosylation at all you can try to express in e.coli but your protein will probably not fold correctly. Insects have both N and O glycosylation. There are enzymes that remove O glycosylation.
If your only problem is heterogeneity of the glycosylation but your protein is otherwise expressed at reasonable levels when secreted I would be tempted to continue with the secreted construct. The fact that it is correctly secreted and the signal peptide cleaved is probably a very good indication that it is correctly folded.
Having said that there are precedents for producing secreted proteins, even functional enzymes and growth factors, as well-expressed soluble cytoplasmic proteins.
Both scenarios are entirely protein- dependent so you cannot really predict what would happen in each case.
Have you tried crystallization of the protein already without removing any sugars or doping the crystal drops with very low levels of PNGase or EndoH/F1?
Thank you, Ulla-Maja,for your information! I do have tried expression in E.coli, which resulted in inclusion body formation. Probably I'll also try point mutations to reduce the glycosylation level. To me the protein homogeneity is very important, for the induction of protein crystallization.
Hi Yutao, inclusion body formation can be regulated and sometimes avoided altogether. There are many threads on RG discussing this problem.
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