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I am reading a patent where they isolate a gene from cDNA constructed from RNA extracted from plant matter.

The subsequent step (in preparation for heterogeneous expression in E Coli.) puzzles me: enter image description here

The codon optimization I can understand but this part confuses me "...remove the first 23 codons from the DNA sequence and replace [the start?] by the ATGGCT sequence."

Why would one remove a set of codons at the start of a sequence? How did they decide how many to remove Is there an obvious reason I am missing? Also what is special about the ATGGCT sequence.

Any ideas why they would do this?

The start of the new sequence (1650 bp) looks like this:

atggctaccg ataatgacag ctctgaaaac cgtcgtatgg gtaattacaa gccgtccatc       60

tggaactacg acttcctgca gtccctggct acccgccaca atatcatgga agagcgccac      120

whereas the original sequence (1710 bp) was this:

atggattctt ccaccgccac cgccatgaga gctccattca ttgatcatac tgatcatgtg       60

aatctcagaa ctgataacga ttcctcagag aatcgaagga tggggaatta taaacccagt      120

Another point I am confused about is that 1710 - 23 x 3 + 6 is 1647. But the new seq. is 1650 bp. What gives?

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    $\begingroup$ Obvious reasons would be: a signal peptide, nope, doesn't look like one, annoying restriction sites, nah, not the case either $\endgroup$
    – Ashafix
    Jun 24, 2016 at 13:23
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    $\begingroup$ Proteolytic cleavage and N-end rule $\endgroup$
    – Ashafix
    Jun 24, 2016 at 15:07
  • $\begingroup$ @Ashafix Can you elaborate more on that? How is the N-end rule applicable in this case? If I translate both those sequences the first Amino Acid is Met. So no stabilizing residue seems to have been added. $\endgroup$ Jul 6, 2016 at 7:32

1 Answer 1

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The easiest answer would be to ask the authors of the patent. Several other possibilities (see comments) were excluded, such as:

  • restriction sites which interfere with cloning
  • signal peptide
  • proteolytic cleavage

Although it's just a theory, removing part of the N-terminus might improve protein stability and half-life time because the first 30-40 amino acids lack an ordered structure (as judged by submitting the structure to Phyre). Those structures are known to aggregate and influence protein stability.

References:

The use of systematic N- and C-terminal deletions to promote production and structural studies of recombinant proteins.

Sequence composition of disordered regions fine-tunes protein half-life

Production of prone-to-aggregate proteins

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