We are trying to express a recombinant protein that is made of two different subunits. We have a plasmid with the alpha subunit and another with the beta subunit, but they are derived from the same plasmid (pcDNA3.1). We want to co-transfect them both at the same time to the cells, but we don't know if using the same promoter sequence for each subunit can impact the expression. Is it better to clone one of the subunits in different empty backbone?
I've co-expressed multiple genes in pCDNA3.1+ plasmids all the time and never had an issue. It might have a slight effect on expression, but it's not likely to have a serious impact, especially if your proteins are compatible and/or dependent on one another. Generally, the much bigger issue is when the activity of one protein impairs the stability or expression of the other.
Note: I mostly used HEK293T cells. It's worth checking the promotor compatibility of your cells as certain lines don't do well with certain promotors, in which case multiple plasmids may cause issues.
Dear Victor Pastre
for antibody recombinant production in mammalian cells, we routinelly co-trasfect the heavy and light chain cloned in 2 vectors with the same backborne and promoter (pcdna3.1 or pcdna 3.4) and both 2 chains are well expressed.
Of course the expression level is not exaclty the same (for mabs if we trasfect the same DNA amout in general the light chain is more expressed than heavy chain) and the expression is also affected by the signal peptide selected for protein secretion in the surnatant. Same gene in the same vector with different signal peptides show very different production level.
best regards
Manuele
Hi there,
2 plasmids with 2 different replication origins is a good point for stability of the transformed strain during growth.
impact
User
Simultaneously expression of two different genes using the same plasmid for each one can impact the expression level
Yes, the simultaneous expression of two different genes using the same plasmid can potentially impact the expression levels of both genes. There are several factors that can influence the expression levels in this scenario:
Promoter Strength: The promoter region of a plasmid plays a crucial role in initiating gene expression. If the two genes are driven by the same promoter, the strength of the promoter may affect the expression levels of both genes. If one gene has a weaker promoter, it may result in lower expression compared to the gene with a stronger promoter.
Gene Size and Composition: The size and composition of the genes can affect their expression levels. Larger genes may require more resources and time for transcription and translation, which could lead to decreased expression levels compared to smaller genes. Additionally, differences in codon usage, secondary structures, or other sequence elements can influence gene expression.
Competition for Resources: The expression of two genes from the same plasmid may lead to competition for cellular resources such as ribosomes, RNA polymerases, or other factors involved in gene expression. If both genes are highly expressed and compete for these resources, it can result in reduced expression levels for both genes.
Gene Interactions: The two genes may interact with each other at the mRNA or protein level, leading to changes in their expression. For example, the presence of one gene product may affect the stability or function of the other gene's mRNA or protein. Such interactions can result in altered expression levels for both genes.
Manirahari Regis With respect to your contribution please consider the following: Interactions of RNA structures from the two genes will be the same whether or not the genes are expressed on a single or two separate plasmids.
High expression vectors can often cause or select for genetic instability of the plasmid or even gene deletion.
I do not understand your argument about the two genes competing with each other for resources. The yield will be determined by rates of initiation of transcription and translation unless there is premature termination of translation where rare tRNA codons are being used in the coding regions and the total rate of synthesis is too high (e.g. after induction?).
Expression of both subunits in the same cell may well be better if the formation of the dimer involves interaction of hydrophobic regions on the two subunits. Separate expression may lead to precipitation of insoluble aggregates which may make purification of the monomer products and their subsequent hetero-dimer difficult.
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