Add like
Add dislike
Add to saved papers

Construction, expression, and characterization of AG1 1-843 and AG1 1-1581 .

AG1, a member of the DUF1220 protein family, exhibits the most extreme human lineage-specific copy number expansion of any protein-coding sequence in the genome. These variations in copy number have been linked to both brain evolution among primates and brain size in humans. Unfortunately, our current understanding of the structure and function of these proteins is limited because current cloning and expression techniques fail to consistently produce recombinant protein for in vitro studies. The present work describes a method for amino acid and DNA sequence optimization and synthesis, recombinant protein expression and analysis of two AG1 fragments, AG11-843 and AG11-1581 . It was first necessary to modify the nucleotide sequence, while holding the GC content at 52.9%. The genes were then sectionally synthesized by overlap PCR. The resulting segments were cloned into the pET-15 b-sumo expression vector and subsequently transformed into BL21 (DE3) cells. After inducing their expression, the AG11-843 and AG11-1581 proteins were isolated and purified. Furthermore, using dynamic light scattering and gel filtration analysis, AG11-843 and AG11-1581 were shown to be present in tetrameric and dimeric forms in solution. To our knowledge, this is the first study to synthesize and express fragments of the DUF1220 protein family for in vitro analysis. Taken together, the proven utility and versatility of this method indicate that it can be used as an effective technique to construct and express other proteins with complicated sequences, thus providing the means to study their function and structure in vitro.

Full text links

We have located links that may give you full text access.
Can't access the paper?
Try logging in through your university/institutional subscription. For a smoother one-click institutional access experience, please use our mobile app.

For the best experience, use the Read mobile app

Mobile app image

Get seemless 1-tap access through your institution/university

For the best experience, use the Read mobile app

All material on this website is protected by copyright, Copyright © 1994-2024 by WebMD LLC.
This website also contains material copyrighted by 3rd parties.

By using this service, you agree to our terms of use and privacy policy.

Your Privacy Choices Toggle icon

You can now claim free CME credits for this literature searchClaim now

Get seemless 1-tap access through your institution/university

For the best experience, use the Read mobile app