1) Yes. They work "OK" - but I prefer to use dedicated vectors mostly for aesthetic reasons, since the TriEx series are kind of bloated (for a good reason). The expression levels are usually alright. So, in conclusion - if you're trying 100 genes in all kinds of systems you could use TriEx (or Gateway, LIC, etc.) but if you have 10 genes to try I would recommend regular vectors.
2) bac-2-bac is a proven, fairly robust system that incorporates an integrity check at an early stage (bacteria). Also you can sequence the prepared bacmid to verify that everything is OK. With the other two systems you're saving a step but (in my opinion) you're reducing your confidence level. With the Gus indicator there's a degree of control but it's a PITA to stain and verify, etc. Direct transfection of pure bacmid is easy, and you get first-passage virus that's almost always good enough to infect massive amounts of cells, so your overall time savings are (in my opinion) greater. 3) STREP tag vs His tag - kind of like comparing a sports car to a lorry. Sometimes you win using one but not the other - however for a generic case I would nearly always prefer the truck. STREP can be essentially one-step, but the yields are often quite poor due to low resin capacity and fragility; the resin is expensive and does not always regenerate well. In comparison, His-tag nearly always works, usually gives loads of protein (as much as your system can express in a competent form), gives adequate purity (if you use His-Select or Talon in combination with detergent lysis you get only a couple of foreign bands) and is cheap to use and easy to regenerate. So, if you're working with something crazy or esoteric - some 250 kDa protein that only expresses in the cerebrospinal fluid of recombinant Madagaskar Chipmunk embryos - then STREP tag is probably better. For an 'easy' protein expressed in bacteria, baculovirus, or yeast - His tag is a reasonable default starting point. Artem -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Anirban Adhikari Sent: Thursday, December 20, 2007 5:56 PM To: [email protected] Subject: [ccp4bb] Question about protein expression (not CCP4 related) Hello, Sorry about the non CCP4 related question. I have few questions regarding protein expression: 1. I was wondering if anyone used the "Multisystem Expression" vectors from Novagen and if so whats your opinion about these vectors? These are the pTriEx series of vectors that can be used for protein expression in E.coli, baculovirus and mammalian cells using the same construct. Since most of the proteins in our lab is expressed in many different systems the pTriEx series of vectors appear particulary useful! 2. We are currently using the Bac to Bac system from Invitrogen for generating baculovirus which requires purification of the bacmid DNA from DH10Bac cells before transfection. Looks like the newer systems, such as BaculoGold (BD biosciences) or BacVector (Novagen) systems skip this step altogether and saves time. Does anyone have experience with both systems and if so which one would you prefer? 3. Is anyone purifying their protein using the Strep-tag II/Strepactin affinity system and if so how does it compare to His-tag? I am looking forward to reading your opinions and suggestions. Thanks a lot, Anirban Anirban Adhikari, Ph. D. Postdoctoral Research Scholar Department of Molecular Biology UT Southwestern Medical Center 5323 Harry Hines Blvd, Dallas, TX 75390-9063
