Michael Stark
Professor Emeritus

The pTTQ18 story

The pTTQ18 story is the story of what, until quite recently, was my most highly cited publication, one of the few research papers on which I am the sole author - something quite unusual these days. There can be no arguments about who did what - I did everything from conceving the idea to correcting the proofs!

I was working as a postdoc in the Leicester Biocentre and was trying unsuccessfully to express recombinant proteins in E. coli for the purpose of raising antibodies. The problem was that the proteins were toxic to the host cell. Because I was using a high-copy vector carrying the lac promoter, it titrated out the low levels of lac repressor from the single chromosomal copy of the lacI gene, leading to high levels of 'uninduced' expression that killed the cells. I realised that the solution was to build a vector that carried its own copy of the lacI gene so that the uninduced levels were rendered negligible.

To build pTTQ18, I bracketed the cloning sites in pUC18 with the strongest available E. coli promoter (the tac promoter developed in the Ptashne lab, a hybrid between the trp and lac promoters) and terminator (the rrnB ribosomal RNA terminator described by Jürgen Brosius) and then added the lacIQ gene from a plasmid constructed by Michele Calos. In 1978, sequencing the lacIQ allele and the finding that it resulted from a single base change in the promoter -35 consensus merited an article in Nature - how things have changed. There were no fancy features like affinity tags and it was all done by standard cloning - PCR hadn't been invented yet. Thus was pTTQ18 born. However, having used it to express my proteins of interest, we completely failed to generate useful antibodies and so much to my chagrin, my only primary research publication deriving from this project is the description of the vector itself. Fortunately, though it has proved very useful for many other groups, leading to the current 279 citations of the original paper. The Rothman lab used it to express NSF, the ATPase that processes cis-SNARE complexes, while in another pTTQ18 has been modified for Gateway cloning and used to express the CorA transporter from Methanosarcina mazei. There have even been some 2015 papers - in one of the latest pTTQ18 has been used for expression of a number of bacterial proteins containing tandem BTP (bacterial transmembrane pair) domains. Word has it that it is particularly useful for expressing bacterial and archaeal membrane proteins such as this one.

pTTQ18 Resources

You can download the pTTQ18 DNA sequence by clicking here. A pdf version of the original Gene paper describing pTTQ18 is also available. Please contact Mike Stark if you would like a sample of pTTQ18.

Word document Gene 51, 255-267 (1987) (pdf document, 2.2 Mb)

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