It's not an irrelevant question and the limits for E. coli from research by others and personal, seems to be

Hi Dr. Shimamoto. Thanks for your insights.  What are the 12kb and 30kb examples of genes introduced with adenoviral vector that you mention? Can  you give me a reference?

But, has anyone actually tried and failed to introduce a 1.5M dalton protein (40kb cDNA) in a mammalian cell or you think there are no reports because nobody has actually ever tried to do so?

Thank you

David.

If a protein that size were successfully produced then it would likely be in the literature unless it is company confidential and therefore restricted from the public due to IP issues. I face those type of issues with my former and current company..  Investigators do attempt to produce single chain proteins of extraordinary sizes and you have probably done some literature searches and found that this is extremely difficult (usually failures).  Usually the strategy is to use hosts that are known to naturally produce proteins of such sizes and therefore eukaryotic systems are used.   Here's an example for both E coli and mammalian:  Microb Biotechnol. 2013 Nov; 6(6): 651–663.

Published online 2013 Oct 11. doi: 10.1111/1751-7915.12081,  PMCID: PMC3815454

NIHMSID: NIHMS516355, Recombinant DNA production of spider silk proteins

Olena Tokareva,1,† Valquíria A Michalczechen-Lacerda,2 Elíbio L Rech,3 and David L Kaplan1,*  Besides expression, do you think it would be straightforward to fold a 1.5 million dalton protein out of E. coli?  

For adenovirus/helper AAV systems try these on for size:  Nature Protocols 2, - 1236 - 1247 (2007), Published online: 17 May 2007 | doi:10.1038/nprot.2007.135, 

Gene Therapy (2016) 23, 86–94; doi:10.1038/gt.2015.75; published online 20 August 2015, Evaluation of helper-dependent canine adenovirus vectors in a 3D human CNS model

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=12&cad=rja&uact=8&ved=0ahUKEwjrlq2D1ZLNAhUP62MKHdH-Dr8QFghrMAs&url=http%3A%2F%2Fwww.amsbio.com%2Fbrochures%2FLentivirus_Brochure_-_AMSBIO.pdf&usg=AFQjCNFsX-jPmalaI-koTDruPDDYPOx25Q&sig2=3RevG1Dei8m5j9lOcrLazw

Dear Dr Shimamoto,

Thank you for the references. I want to try to express in mammalian system like CHO or HEK.

Hi Dr. Shimamoto,

I have now read the references you sent. So, I see the adenovirus vectors deal with inserting into the cells plasmids of about 33kb...41kb is not that far...So it seams that at least the problem might not be at the transfection level...

Then the silk proteins business, its completely a different game, because although they say silk is like 3500aa long, its actually a hetero-polymeric entity and to work, multiple proteins need to come together, etc...so its not like expression one single giant 1.5M dalton protein and be it...it seems more tricky and long...protein ratios, amounts, purification, etc..

So in the end my initial question remains totally unanswered....Maybe it doesn't fold, maybe doesn't this or that...but no clear cut answer like " you are gonna hit a brick wall because of this or that"...

Thank you anyways

d.

Hi Dr. Shimamoto,

just to followup on your type of advise. The first brick wall I am finding is dealing with the 40kbp construct to subclone...Does anyone know who can create and subclone 40,000 bp construct DNA?

Thank you

David.

If your literature literature search is difficult and others have not provided any examples then it might be because the nature of the protein needs to be described in order to determine feasibility.  In other words there are many aspects to consider to determine what may go right/wrong and these aspects depend upon the nature of this single polypeptide.  For example:  post-translational modification, cell toxicity, pre-pro forms, secretion, cytosolic or membranous, enzyme or structural, artificial or natural, function, degradation, aggregation, etc. The amino acid sequence would be the most useful at this point for a definitive answer from a protein chemistry perspective. A short motif continuously repeated up to 1.5 million daltons might be the simplest case. 

Dear Dr. Shimamoto,

Thank you for your kind reply. But again, is of little help. So far, I have already discovered the the first brick wall and that is that its not possible to synthesize the 41kb DNA construct to express the 1.5M dalton protein. And even if its done in pieces, subcloning them into the final 41kb construct is probabilistic...unknown if it can be done. Any thoughts on how to assemble or synthesize a 41kb construct?

Thank you

David.

You would have to disclose a relatively large amount of information to determine the assembly/synthesis strategy because this is a very specific case.  Therefore there may not be any way to help out at this point.