both I27<sup>RS</sup>₈ and a variant I27<sup>GLG</sup>₁₂. Their
I27<sup>RS</sup>₈ procedure is:
-* Human cardiac muscle used to generate a cDNA library [Rief 1997]
-* cDNA library amplified with PCR
+* Human cardiac muscle used to generate a [cDNA][] library ([Rief 1997][r97])
+* cDNA library amplified with [PCR][]
- 5' primer contained a BamHI restriction site that permitted
- in-frame cloning of the monomer into the expression vector pQE30
- (Qiagen, Chatsworth, CA).
+ in-frame cloning of the monomer into the expression vector pQE30.
- The 3' primer contained a BglII restriction site, two Cys codons
located 3' to the BglII site and in-frame with the I27 domain,
and two in-frame stop codons.
They also give the full-length sequence of I27<sup>RS</sup>₈:
- Met-Arg-Gly-Ser-(His)₆-Gly-Ser-(I27-Arg-Ser)₇-I27-...-Cys-Cys
+ Met-Arg-Gly-Ser-(His)₆-Gly-Ser-(I27-Arg-Ser)₇-I27-...-Cys-Cys
-They point out the Arg-Ser (RS) amino acid sequence is the BamHI-BglII
-hybrid site, which makes sense (see below).
+They point out the Arg-Ser (RS) amino acid sequence is the BglII/BamHI
+hybrid site, [which makes sense](#BglII-BamHI-joint).
Back on the Athena site, they have a [page describing their
procedure][I27O-syn] (they reference the Carrion-Vazquez paper). They
In their note 11, Rief et al. explain their synthesis procedure:
-* λ cDNA library
+* [λ][] cDNA library
* Titin fragments of interest were amplified by PCR
* cloned into pET 9d
-* NH₂-terminal domain boundaries were as in [Politou 1996].
+* NH₂-terminal domain boundaries were as in [Politou 1996][p96].
* The clones were fused with an NH₂-terminal His₆ tag and a
COOH-terminal Cys₂ tag for immobilization on solid surfaces.
online sources for pHK414. Kempe cites [R.J. Watson et al.
*Expression of Herpes simplex virus type 1 and type 2 glyco-protein D
genes using the Escherichia coli lac promoter.* Y. Becker (Ed.),
-*Recombinant DNA Research and Viruses*. Nijhoff, The Hague, 1985,
+*Recombinant DNA Research and Viruses.* Nijhoff, The Hague, 1985,
pp. 327-352.][w85])
### Synthetic SP
#### pHK414 + HindIII + BamHI
+They cut a hole in the plasmid…
+
HindIII BamHI.
(PstI) BglII,| |
CTGCAG...AGATCTA GATCCAAGATCC
#### SP + HindIII + BamHI
+… and cut matching snips off their SP gene.
+
HindIII. ,BamHI_.
| | Met Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met |
AGC TTC ATG CGT CCG AAG CCG CAG CAG TTC TTC GGT CTC ATG
#### pSP4-1
+Mixing the snips together gives the plasmid with a single SP.
+
HindIII BamHI.
,PstI. BglII.| | MetArgProLysProGlnGlnPhePheGlyLeuMet |
CTGCAG...AGATCTAAGCTTCATGCGTCCGAAGCCGCAGCAGTTCTTCGGTCTCATGGATCCAAGATCC
### Synthesizing pSP4-2
-pSP4-1 is split in two parallel rections
+The single-SP plasmid, pSP4-1, is split in two parallel reactions.
#### PstI + BamHI
Like Kempe, Carrion-Vazquez et al. flank the I27 gene with BglII and
BamHI, but they reverse the order. Here's the output of their PCR:
- BamHI-I27-BglII-Cys-Cys-STOP-STOP
+ BamHI-I27-BglII-Cys-Cys-STOP-STOP
From the PDB entry for I27 ([1TIT][]), the amino acid sequence is:
nucleotides 115 through 203 is:
,RGS-His epitope__________________. ,BamHI.
- Met Arg Gly Ser His His His His His His Gly Ser Ala Cys Glu Leu ...
- ATG AGA GGA TCG CAT CAC CAT CAC CAT CAC GGA TCC GCA TGC GAG CTC ...
- CGT CTC TTC GAT ACG ACA ACG ACA ACG ACA TTC GAA TAC GTA TCT AGA ...
+ Met Arg Gly Ser His His His His His His Gly Ser Ala Cys Glu Leu
+ ATG AGA GGA TCG CAT CAC CAT CAC CAT CAC GGA TCC GCA TGC GAG CTC
+ CGT CTC TTC GAT ACG ACA ACG ACA ACG ACA TTC GAA TAC GTA TCT AGA
,SmaI__.
,KpnI_. HindIII
sequence? If there is, why do Carrion-Vazquez et al. not
acknowledge it when they write [3]:
- The full-length construct, I27<sup>RS</sup>₈, results in the following
- amino acid additions: (i) the amino-terminal sequence is
- Met-Arg-Gly-Ser-(His)6-Gly-Ser-I27 codons; (ii) the junction
- between the domains (BamHI-BglII hybrid site) is Arg-Ser; and
- (iii) the protein terminates in Cys-Cys.
+ > The full-length construct, I27<sup>RS</sup>₈, results in the
+ > following amino acid additions: (i) the amino-terminal sequence is
+ > Met-Arg-Gly-Ser-(His)6-Gly-Ser-I27 codons; (ii) the junction
+ > between the domains (BamHI-BglII hybrid site) is Arg-Ser; and
+ > (iii) the protein terminates in Cys-Cys.
Since they don't acknowledge an I27-Arg-Ser-Cys-Cys ending, might
there be more amino acids in the C terminal addition?
Since I'm stuck trying to get I27 into either plasmid, let's try and
work backward from
- Met-Arg-Gly-Ser-(His)₆-Gly-Ser-(I27-Arg-Ser)₇-I27-Cys-Cys
+ Met-Arg-Gly-Ser-(His)₆-Gly-Ser-(I27-Arg-Ser)₇-I27-Cys-Cys
+
+#### <a id="BglII-BamHI-joint">BglII/BamHI joint</a>
The BglII/BamHI overlap would produce the expected Arg-Ser joint.
#### Continuing to the first plasmid, pI27-1 must have been
- remote ... ,RGS-His epitope__________________. ,BamHI. I27...
- ... Met Arg Gly Ser His His His His His His Gly Ser Leu Ile ...
- ??? ... ATG AGA GGA TCG CAT CAC CAT CAC CAT CAC GGA TCC CTA ATA ...
- ??? ... CGT CTC TTC GAT ACG ACA ACG ACA ACG ACA TTC GAA GAT TAT ...
+ remote ... ,RGS-His epitope__________________. ,BamHI. I27...
+ ... Met Arg Gly Ser His His His His His His Gly Ser Leu Ile ...
+ ??? ... ATG AGA GGA TCG CAT CAC CAT CAC CAT CAC GGA TCC CTA ATA ...
+ ??? ... CGT CTC TTC GAT ACG ACA ACG ACA ACG ACA TTC GAA GAT TAT ...
- ........I27 ,BglII. continuation of pQE30?
- ... Glu Leu Arg Ser Cys Cys STOPSTOP...
- ... GAA TTG AGA TCT TGC TGC TAG TAG ...
- ... CTT AAC CTC GAG GTA GTA GCT GCT ...
+ ........I27 ,BglII. continuation of pQE30?
+ ... Glu Leu Arg Ser Cys Cys STOPSTOP...
+ ... GAA TTG AGA TCT TGC TGC TAG TAG ...
+ ... CTT AAC CTC GAG GTA GTA GCT GCT ...
### Potential pQE30 insertion points
[cv99]: http://dx.doi.org/10.1073/pnas.96.7.3694
+[r97]: http://dx.doi.org/10.1126/science.276.5315.1109
+[PCR]: http://en.wikipedia.org/wiki/Polymerase_chain_reaction
+[cDNA]: http://en.wikipedia.org/wiki/Complementary_DNA
+[λ]: http://en.wikipedia.org/wiki/Lambda_phage
[AthenaES]: http://www.athenaes.com/
[I27O]: http://www.athenaes.com/I27OAFMReferenceProtein.php
[I27O-tb]: http://www.athenaes.com/tech_brief_I27O_protein.php
[I27O-syn]: http://www.athenaes.com/Projects_Polyproteins.php
[k85]: http://dx.doi.org/10.1016/0378-1119(85)90318-X
+[p96]: http://dx.doi.org/10.1006/jmbi.1996.0050
[gcode]: http://en.wikipedia.org/wiki/Genetic_code
[renz]: http://en.wikipedia.org/wiki/Restriction_enzyme
[BamHI]: http://en.wikipedia.org/wiki/BamHI
[w85]: http://books.google.com/books?id=eA6iSmR0I4wC
[1TIT]: http://www.pdb.org/pdb/explore/explore.do?structureId=1TIT
[NM_003319.4]: http://www.ncbi.nlm.nih.gov/nuccore/NM_003319
-[Q8WZ42] http://www.uniprot.org/blast/?about=Q8WZ42[12677-12765]
-[var] http://web.expasy.org/cgi-bin/variant_pages/get-sprot-variant.pl?VAR_040140
-[pQE30-a] http://www.qiagen.com/literature/vectors_pqe.aspx
-[pQE30-b] http://www.qiagen.com/literature/pqesequences/pqe-30w.txt
-[pUC19-a] http://bccm.belspo.be/db/lmbp_plasmid_details.php?NM=pUC19
-[pUC19-b] http://www.ncbi.nlm.nih.gov/nucleotide/M77789?report=genbank
+[Q8WZ42]: http://www.uniprot.org/blast/?about=Q8WZ42[12677-12765]
+[var]: http://web.expasy.org/cgi-bin/variant_pages/get-sprot-variant.pl?VAR_040140
+[pQE30-a]: http://www.qiagen.com/literature/vectors_pqe.aspx
+[pQE30-b]: http://www.qiagen.com/literature/pqesequences/pqe-30w.txt
+[pUC19-a]: http://bccm.belspo.be/db/lmbp_plasmid_details.php?NM=pUC19
+[pUC19-b]: http://www.ncbi.nlm.nih.gov/nucleotide/M77789?report=genbank
[[!tag tags/theory]]
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