One possibility is that the 2nd band is a contaminant because purification is incomplete. Getting rid of it will require a modification of the purification procedure, such as adding another step.

Another possibility is that the protein is a heterodimer, i.e. it consists of 2 different subunits with different molecular weights.

A third possibility is that the lower molecular weight band is a product of proteolysis. Taking more care during purification to prevent proteolysis, and including inhibitors of proteolysis during purification, may help to reduce this problem.

Another possibility is that the two bands represent low and high glycosylation forms of the same protein. You can test this by treating the protein with PNGase F, which removes N-linked glycosylation.

Thanks a lot dear @Adam for all the possibilities you mentioned. There are some points I should add:

- Lentil lectin chromatography itself is the second step of hemagglutinin purification which is done after ion exchange chromatography. It's usually the final purification step of glycoproteins.

- Hemagglutinin is a homotrimer protein so we expect te see a single bond.

In my opinion, low/high glycosylation and/or proteolysis are the strongest possibilities.

- How we can test it with PNGase F treatment and what are the expected results?

- What is the the percentage of deglycosylation (high to low glycosylation) during the affinity chromatography step?

Another possibility occurred to me: Could the other band be lentil lectin that is coming off the resin? This once happened to me with RCA lectin resin.

If a difference in N-linked glycosylation is the issue, treating the sample with PNGase F, which removes N-linked glycosylation, should cause the two bands to become one.

I worked with a glycosylated membrane protein that ran as two broad bands that differed in the level of glycosylation. I think this difference occurred during biosynthesis, rather than being an artifact of purification.

Thanks so much dear @Adam.

Since the protein of second bond is about 65 kDa and lentil lectin is a 46 kDa protein, I think the bond belongs to hemagglutinin.

On the other hand, we see the two bonds after ion exchange chromatography as well, before starting lentil lectin.

I think we need to test the treatment with PNGase F as well as adding a protease inhibitor to see if rest bonds, around 20-30 kDa, are removed.

What system are using using to produce the protein (bacteria, yeast, insect, mammalian etc)?

Hello @Samuel Davis,

Our expression host is Baculovirus-Insect (SF9) Cells

Hadi Nedaei I suspected that you were using insect cells, as incomplete glycosylation is a common problem with baculovirus infected insect cells. This appears as a characteristic double band, like shown on your gel, and is likely because the cellular machinery is overwhelmed by the excessive amount of mRNA transcribed by the polyhedrin promoter most commonly used by baculovirus vectors, so protein processing and quality control are compromised (plus the effect of the secretory pathway being stressed by the viral infection and being hijacked for virus replication).

This was a big problem for me during my PhD when I was recombinantly producing membrane proteins. I was eventually able to solve the problem though by using the "EarlyBac system" developed by the lab of Prof. Hiro Furukawa at Cold Spring Harbour. Put shortly, this is a series of vectors that they developed using an early DmHsp70 promoter. While this promoter is weaker and gives overall slightly lower levels of total protein, as it transcribes less mRNA it puts less stress on the secretory pathway and overall the protein produced is of higher quality. Although developed for use with membrane proteins, it can also be used with soluble proteins.

If you are using the Bac-to-Bac system to make your baculovirus, the restriction sites on the vector are identical to those on the commonly used pFastBac1 vector, so you can just drop your gene in. There are more details on the EarlyBac system in the paper at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8896536/ and the Furukawa lab have a useful page as well on using it at https://furukawalab.labsites.cshl.edu/protocols/earlybac/

You can get the vector from Addgene at https://www.addgene.org/170460/

As an example, I've attached a quick figure I've made showing two gels from my PhD thesis comparing the standard pFastBac1 vector to the EarlyBac vector. You can immediately see the difference in the Earlybac system giving only a single band throughout with everything being correctly glycosylated. If you want more detail, feel free to look in my thesis, the stuff of interest is in Chapter 5. https://discovery.dundee.ac.uk/en/studentTheses/recombinant-production-and-investigation-of-neurotransmitter-chan

If glycosylation is not essential to your proteins activity though, you could just digest the protein with PNGase F to remove the glycans, which will yield a single product with no attached glycans. However, using PNGase F under non-denaturing conditions to retain functional protein is a lot more difficult than just using it under denaturing conditions to run on a gel etc.

Dear Samuel Davis ,

Thank you so much for your great and comprehensive answer, suggestions, and help. Since we should use lentil lectin resin instead of Streptactin-XT, and it captures only glycosylated forms of proteins, the double bond would most likely be due to low and high glycosylation of hemagglutinin. If this is the case and cellular machinery is overwhelmed by too much mRNA transcribed, I don't know why only two bonds appear without any additional bonds in between.

You may confirm whether the second band is hemagglutinin or not by employing mass spec or Western analysis. If the result comes as positive then it indicates the glycosylation differentiation ended as varying MW. Deglycosylation is a good approach to confirm this hypothesis. If you need to obtain a single form (as a purification target) you should add 3rd step to your purification, SEC for instance. IEX and glycoprotein affinity chromatography combined is not sufficient to get a single band as you experienced. This may also be an inefficient fractionation window problem during protein purification (FPLC?). Different glycosylation should result in separated protein responses (peaks) at the LC system. Chromatographic resolution can be improved by shallower gradient applications and differently glycosylated proteins may be well separated and isolated. Using Boronate affinity or preferring another elution strategy can also enhance the resolution efficiency at glycoprotein affinity purification. If all these are not possible, you may integrate the SEC application as 3rd step of tandem purification to get a single band.

Dear @ismail Emir Akyildiz

Thanks so much for your very helpful answer. We will be trying SEC method and Boronate affinity chromatography. Using lentil lectin, we only see a single peak in the FPLC chromatogram, while a double bond on the gel.