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           RFC 14: Protein Domain Fusions in BB-2 Assembly
                          November 23, 2008
                              Tom Knight

Related RFCs: 9, 11, 12, 13

Keywords: BB-2 assembly, protein fusions, protein tags


Original Biobrick assembly scars left out-of-frame assembly, and when in
fame, left poor amino acid choices for protein fusions.  With the BB-2
assembly standard, protein fusions are easy and leave an in-frame
Ala-Ser scar.  This provides us with the opportunity to rethink the
partitioning of protein coding regions, with a much greater emphasis
on the modularity of the protein structure.  In particular, it now is
desirable to think about N terminal domains such as export tags, C
terminal domains such as degradation tags, and both N and C terminal
tags and cleavage sites.

RFC 13 describes a proposed fragmentation of coding regions into a Head,
zero or more Domains, and a Tail region.  The scar linking these
regions is undefined there, but here we consider the specific case of
BB-2 scars, with sequence GCTAGT, coding for the amino acid sequence


Specifically, we propose here a rethink of the RBS/CDS region,
resulting in a fusion of the ribosomal binding site with the start
codon.  A typical RBS/CDS start then would consist of an RBS, a spacer
to the start codon, followed by an ATG start codon.  The assembly of
this RBS/Start with a protein Domain would result in an RBS and the N terminal
coding region consisting of Met-Ala-Ser, followed by the Domain.  This
standardizes the initial few amino acids of each coding region, in
combination with the ribosomal binding site, as a way of creating more
standard translation levels.  It is well known that the first few
codons have significant effect on translation efficiency, so
standardizing those, along with the RBS, should result in less
expression variability.  This standardization could not be performed
along with protein export tags, but could, in most cases, be used with
N terrminal tags.

In BB-2 assembly, The translation efficiency of the Met-Ala-Ser N
terminal region (with the specified codons) is approximately 60%
compared to the best efficiency initiation, with the sequence
AUGAAA (Met-Lys) (Looman87, Stenstrom01, Stenstrom01a).

For proteins needing custom N terminal codons, such as export signal
tags, these can be constructed as custom RBS/Head domains.  See
(Zalucki07, Zalucki07a, Zalcki08) for a discussion of codon usage in
export tag leaders.

Note that the initial f-Met in coding regions constructed with BB-2
assembly is highly susceptible to pratease cleavage with the
formyl-methionine-peptidase protein (map) in E. coli (see Frottin06),
so mature proteins would have an N-terminal alanine.

More specifically, I propose a re-worked set of Head RBS + ATG components
in the registry, consisting of many of the existing RBS components,
using the BB-1 scar as the RBS-ATG spanning sequence.  A typical
RBS+ATG part then might have the sequence AGGAGGACTAGATG with the BB-1
mixed site preceeding the ATG start codon.  This would retain some
consistency of sequence with the existing RBS measurements, while
moving to the new BB-2 standard.

Existing protein coding regions would be reworked to remove the
initial start codon, stop codon, and any tags or degradation tails.

A new set of Tail parts, consisting of TAATAA stops would be
constructed.  Additionally, a set of E. coli degradation tails of
different lifetimes would be made, including the TAATAA stop codon

A new set of protein fustion tags would also be made.  A partial list
includes this set (partially from the excellent resource in the tables
of Kimple04):

             protein purification tags, antibody epitopes

         pmid 8770418



        (should this really be 7His? Interspersed AA for patent reasons?)


GST (n-terminal)

Chitin binding

BCCP biotin carboxyl carrier protein
        PMID: 14736427
        PMID: 10470036
	PMID: 10211839


Thrombin cleavage domain
         LVPRGS (Leu-Val-Pro-Arg-Gly-Ser), factor Xa cleaved

Calmodulin tag


GFP protein tags

maltose binding protein


protein splicing domains (inteins)

surface display proteins
        Neisseria IgA1

Frottin F, Martinez A, Peynot P, Mitra S, Holz RC, Giglione C, Meinnel
T.  The proteomics of N-terminal methionine cleavage.  Mol Cell
Proteomics. 2006 Dec;5(12):2336-49. Epub 2006 Sep 8.  PMID: 16963780

Kimple ME, Sondek J.  Overview of affinity tags for protein
purification.  Curr Protoc Protein Sci. 2004 Sep;Chapter 9:Unit
9.9. Review.  PMID: 18429272

Looman AC, Bodlaender J, Comstock LJ, Eaton D, Jhurani P, de Boer HA,
van Knippenberg PH.  Influence of the codon following the AUG
initiation codon on the expression of a modified lacZ gene in
Escherichia coli.  EMBO J. 1987 Aug;6(8):2489-92.  PMID: 3311730

Stenstrom CM, Jin H, Major LL, Tate WP, Isaksson LA.  Codon bias
at the 3'-side of the initiation codon is correlated with translation
initiation efficiency in Escherichia coli.  Gene. 2001 Jan
24;263(1-2):273-84.  PMID: 11223267

Stenstrom CM, Holmgren E, Isaksson LA.  Cooperative effects by the
initiation codon and its flanking regions on translation initiation.
Gene. 2001 Aug 8;273(2):259-65.  PMID: 11595172

Zalucki YM, Power PM, Jennings MP.  Selection for efficient
translation initiation biases codon usage at second amino acid
position in secretory proteins.  Nucleic Acids
Res. 2007;35(17):5748-54.  PMID: 17717002

Zalucki YM, Jennings MP.  Experimental confirmation of a key role for
non-optimal codons in protein export.  Biochem Biophys Res
Commun. 2007 Mar 30;355(1):143-8. Epub 2007 Jan 31.  PMID: 17291454

ExPASy PeptideCutter: The cleavage specificities of selected enzymes and chemicals