Axel Knebel receives CLS Innovator of the Year Award for developing a new protein affinity purification tag
Many congratulation to Axel Knebel who has just received the CLS Innovator of the Year Prize for the development of a novel affinity purification tag termed the 'Dac-tag'.
Axel, runs the MRC-PPU Protein Production and Assay Development in the ubiquitylation system team that generate the majority of ubiquitylation reagents that the MRC-PPU researchers and their collaborators depend on.
Axel developed the Dac-tag in order to greatly facilitate the purification of recombinant proteins from bacteria or insect cells. Using this methodology Axel and his team have been able to obtain incredibly pure preparations of enzymes especially from insect cell expression systems to a much greater degree than could be achieved with other tags such as His, maltose binding and GST. The Dac-tag is now becoming the frontline choice for the affinity purification of recombinant proteins from insect cells. It also has benefits as it is a monomeric and highly soluble protein that helps stabilise and enable better expression of recombinant proteins.
The Dac-tag is based on a fragment of penicillin binding protein 5 (PBP5 residues 37-297), which binds in a pseudo reversible manner to ampicillin Sepharose. In order to obtain a pure protein of interest, the Dag-tag is cloned to the N or C-terminal end of the protein. Because of the properties of the Dac-tag, this fusion protein can then be readily isolated in a highly purified manner using ampicillin Sepharose, which is very selective for penicillin binding proteins. The Dac-tag has the advantage that it is monomeric and does not rely on cysteine chemistry or metal affinity, so it is ideal for purification of proteins in the Ubiquitin arena. Ampicillin or amoxycillin, coupled to NHS-activated Sepharose provide high capacity. Any protein fused to the PBP5 residues 37-297 will rapidly bind to such a Sepharose and can be eluted in a buffer system containing 10mM ampicillin and 5% glycerol. Without these eluents the tag binds tightly and a protein can be recovered for example by using a protease site between the tag and the target protein.
To read the paper describing the development and use of the Dac Tag click here. Examples of the proteins that Axel has purified using this system include the Cullin-1 CDC53/Hrt1 complex, the Cul5/RNF7 and APPBP1/UBA3 complexes as well as the Cullin3-SKP1-KLHL3 complex that plays a critical role in regulating human blood pressure.
The Dac-tag is now being marketed by Expedeon Ltd under the name ‘Cool-tag'. Axel Knebel stated "I am very pleased to receive CLS Innovator of the Year Award for developing a the Dac-tag. I hope that it will help researchers to more easily isolate highly purified recombinant proteins and make a significant contribution to Medical Research. I am delighted to see this tag being widely utilised within the MRC-PPU and hope that its use will be quickly adopted by other researchers'.
Axel is the second MRC-PPU researcher to be awarded the CLS Innovator award the first being Ayaz Najafov a PhD student at the time in Dario Alessi's lab who developed the Protein Guru programme to help analyse mass spectrometry data.
Axel, runs the MRC-PPU Protein Production and Assay Development in the ubiquitylation system team that generate the majority of ubiquitylation reagents that the MRC-PPU researchers and their collaborators depend on.
Axel developed the Dac-tag in order to greatly facilitate the purification of recombinant proteins from bacteria or insect cells. Using this methodology Axel and his team have been able to obtain incredibly pure preparations of enzymes especially from insect cell expression systems to a much greater degree than could be achieved with other tags such as His, maltose binding and GST. The Dac-tag is now becoming the frontline choice for the affinity purification of recombinant proteins from insect cells. It also has benefits as it is a monomeric and highly soluble protein that helps stabilise and enable better expression of recombinant proteins.
The Dac-tag is based on a fragment of penicillin binding protein 5 (PBP5 residues 37-297), which binds in a pseudo reversible manner to ampicillin Sepharose. In order to obtain a pure protein of interest, the Dag-tag is cloned to the N or C-terminal end of the protein. Because of the properties of the Dac-tag, this fusion protein can then be readily isolated in a highly purified manner using ampicillin Sepharose, which is very selective for penicillin binding proteins. The Dac-tag has the advantage that it is monomeric and does not rely on cysteine chemistry or metal affinity, so it is ideal for purification of proteins in the Ubiquitin arena. Ampicillin or amoxycillin, coupled to NHS-activated Sepharose provide high capacity. Any protein fused to the PBP5 residues 37-297 will rapidly bind to such a Sepharose and can be eluted in a buffer system containing 10mM ampicillin and 5% glycerol. Without these eluents the tag binds tightly and a protein can be recovered for example by using a protease site between the tag and the target protein.
To read the paper describing the development and use of the Dac Tag click here. Examples of the proteins that Axel has purified using this system include the Cullin-1 CDC53/Hrt1 complex, the Cul5/RNF7 and APPBP1/UBA3 complexes as well as the Cullin3-SKP1-KLHL3 complex that plays a critical role in regulating human blood pressure.
The Dac-tag is now being marketed by Expedeon Ltd under the name ‘Cool-tag'. Axel Knebel stated "I am very pleased to receive CLS Innovator of the Year Award for developing a the Dac-tag. I hope that it will help researchers to more easily isolate highly purified recombinant proteins and make a significant contribution to Medical Research. I am delighted to see this tag being widely utilised within the MRC-PPU and hope that its use will be quickly adopted by other researchers'.
Axel is the second MRC-PPU researcher to be awarded the CLS Innovator award the first being Ayaz Najafov a PhD student at the time in Dario Alessi's lab who developed the Protein Guru programme to help analyse mass spectrometry data.