I am dealing with a strange problem. I want to overexpress PIK3R4 protein. I have 2 different constructs. The first construct encodes naive protein with Puromycin resistance gene. I transfect this construct to HEK cells but do not detect any increase in protein level neither in transient transfection nor after stable Puromycin selection. I thought it could be an antibody issue. But I have a second construct which encodes Myc tagged PIK3R4 with an IRES and GFP. After transfection I can observe GFP expression indicating that there is transcription. But I can not detect an incresase in PIK3R4 protein levels as assesed by PIK3R4 or Myc tag antibodies. What could be the culprit?
Just as a response to some of the suggestions below:
- I have verified the sequence of my plasmid by Sanger sequencing.
- The myc tag is in frame with the protein.
- There is no problem with the KOZAK sequence.
- PIK3R4 is not a secreted protein.
- Plasmid contains CMV promoter and I can see GFP expression indicating effective transcription as GFP is expressed from the same transcript with IRES element.
Thanks in advance
One suggestion is to sequence your plasmids and look if everything is in-frame. You may have thought about it, But any-case try another cell line like 293T which is more robust for transient protein expression.
I have a list of trouble shooting suggestion:
1. Check whether PIK3R4 is secreted in medium.If yes, ELISA could be used to detect it.
2. May be promoter driving PIK3R4 has undergone epigenetic silencing. Clone the gene in different vector containing strong promoter like CAG or CMV
Best
either the plasmid (can sequence it to confirm), or delivery (but looks like it works- based on another plasmid), or detection of protein (i'd look into this- use controls). also start fresh- new cells, media and everything
Are you sure that the CMV promoter is not mutated and functional? As you can see the GFP you don't have delivery problems and the IRES is doing its job. But maybe you have some problem due to the inactivation of the promoter (or the frame but you already checked it). Maybe you can try to use another bacteria strain, purify again and see what happens (I'm not sure it will help but you can try). And check again frame, sequence and possible stops in the coding sequence. Hope it helps.
If CMV promoter is mutated, we sholdnt be able to see GFP expression too, as CMV is the promoter for both GFP and PIK3R4.
Amended on 1 March 2018
Fetal hepatocyte Hc has no HHV-5 (CMV). Hepatoma HepG2 (cultured without fucoidan ) has it at 2.7 μg/mg of cell protein. Fucoidan reduces it to 1.9 μg/mg of cell protein, or reduces for 30% in three days at 0.102 mg/mL (please see file; HepG2 fucoidan). Complete reduction of HHV-5 may be necessary for three weeks.
Thus, endogeneous HHV-5 may influence the experiments.
By the way, PIK3R4/PI3-kinase p150 subunit/Phosphoinositide 3-kinase regulatory subunit 4 has not been detected in my specimens (Hc, HepG2, and 5 liver tissues).
Only LC tissue (named as No.6) has expressed PIK3R1/Phosphatidylinositol 3-kinase regulatory alpha subunit/PI3K regulatory subunit alpha at 6.8 μg/mg of tissue protein. Since gene expression is under the control of virus, I have searched for the culprit in this tissue. Genome polyprotein (Louping ill virus/LIV) is present at 6.4 μg/mg of cell protein in this unique Japanese liver-tissue.
Therefore, the rare protein of PIK3R4 may be difficult to be expressed in the human cells especially absent of Louping ill virus/LIV. My protein database suggests that fetal cells (Hc and may be HEK) have fewer infected-virus as compared to adult-derived cell lines (please see file again; HepG2 fucoidan).
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