From: W. Trevor King Date: Wed, 3 Aug 2011 00:08:27 +0000 (-0400) Subject: Markdown cleanups to I27-synthesis post. X-Git-Url: http://git.tremily.us/?a=commitdiff_plain;h=60fa2ec45c51b493e26b8ff5f8b42fc1d050faf1;p=blog.git Markdown cleanups to I27-synthesis post. --- diff --git a/posts/I27-synthesis.mdwn b/posts/I27-synthesis.mdwn index f56ba95..82537c3 100644 --- a/posts/I27-synthesis.mdwn +++ b/posts/I27-synthesis.mdwn @@ -14,11 +14,10 @@ corresponds to Carrion-Vazquez's I27RS₈. In both I27RS₈ and a variant I27GLG₁₂. Their I27RS₈ 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. @@ -33,10 +32,10 @@ I27RS₈ procedure is: They also give the full-length sequence of I27RS₈: - 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 @@ -53,10 +52,10 @@ Rief 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. @@ -135,7 +134,7 @@ Inserted their poly-SP into pHK414 (I haven't been able to find any 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 @@ -160,6 +159,8 @@ pp. 327-352.][w85]) #### pHK414 + HindIII + BamHI +They cut a hole in the plasmid… + HindIII BamHI. (PstI) BglII,| | CTGCAG...AGATCTA GATCCAAGATCC @@ -169,6 +170,8 @@ pp. 327-352.][w85]) #### 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 @@ -176,6 +179,8 @@ pp. 327-352.][w85]) #### pSP4-1 +Mixing the snips together gives the plasmid with a single SP. + HindIII BamHI. ,PstI. BglII.| | MetArgProLysProGlnGlnPhePheGlyLeuMet | CTGCAG...AGATCTAAGCTTCATGCGTCCGAAGCCGCAGCAGTTCTTCGGTCTCATGGATCCAAGATCC @@ -194,7 +199,7 @@ terminal G, which is part of the BamHI match sequence). ### 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 @@ -239,7 +244,7 @@ I27RS₈ procedure 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: @@ -281,9 +286,9 @@ From the [Qiagen site][pQE30], the section around the linker 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 @@ -327,11 +332,11 @@ need to use a third restiction enzyme to insert their I27. sequence? If there is, why do Carrion-Vazquez et al. not acknowledge it when they write [3]: - The full-length construct, I27RS₈, 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, I27RS₈, 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? @@ -341,7 +346,9 @@ need to use a third restiction enzyme to insert their I27. 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 + +#### BglII/BamHI joint The BglII/BamHI overlap would produce the expected Arg-Ser joint. @@ -421,15 +428,15 @@ so we'll assume the final plasmid was: #### 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 @@ -442,11 +449,16 @@ so we'll assume the final plasmid was: [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 @@ -459,11 +471,11 @@ so we'll assume the final plasmid was: [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]]