Advertisement
RayBiotech
RayBiotech

Inclusion Bodies Be Gone

Scientists routinely express heterologous proteins in Escherichia coli. However, these proteins may not fold properly in the bacteria, causing them to aggregate and form insoluble inclusion bodies. Scientists traditionally denature these inclusion bodies with either 8 M urea or 6 M guanidine hydrochloride to solubilize them. These proteins must then be renatured by replacing the denaturants with a gentler buffer. Unfortunately, this process is highly empirical and laborious, and it often results

By | June 25, 2001

Scientists routinely express heterologous proteins in Escherichia coli. However, these proteins may not fold properly in the bacteria, causing them to aggregate and form insoluble inclusion bodies. Scientists traditionally denature these inclusion bodies with either 8 M urea or 6 M guanidine hydrochloride to solubilize them. These proteins must then be renatured by replacing the denaturants with a gentler buffer. Unfortunately, this process is highly empirical and laborious, and it often results in poor yields. In June 2000, Madison, Wis.-based Novagen Inc. launched the pET 43.1 vector series, specifically to overcome the inclusion body problem. These vectors encode an N-terminal moiety (the Nus·Tag™ sequence) that greatly enhances the solubility of proteins expressed as fusions with the tag. In fact, the polypeptide encoded by the nusA gene (54.8 kDa, 495 amino acids) was carefully selected from more than 4,000 E. coli protein sequences, for having the greatest solubility potential.

Researchers at Novagen employed a solubility model developed by D.L. Wilkinson and R.G. Harrison to identify E. coli proteins likely to confer the highest solubility on a fusion partner.1 They then narrowed the range of choices based on size and published data, to end up with three candidate proteins: NusA, bacterioferritin (BFR), and GrpE. Western blot and SDS-PAGE analyses revealed that NusA fusion proteins were more soluble than were fusions with either BFR or GrpE, or with thioredoxin (TRX) and glutathione-S-transferase (GST), two proteins traditionally used to increase solubility of target proteins. A 54-kDa tyrosinase was efficiently expressed as a NusA fusion, suggesting that the Nus·Tag may be a good carrier for large, insoluble proteins.

According to Mark Lepinske, marketing communications manager at Novagen, "The primary feature of the new Novagen Nus·Tag System is the tremendous solubility this new tag tends to confer on a target protein. I don't know of another fusion tag for solubility that performs better in lab tests. The peer-reviewed literature on this technology is very compelling."

Novagen's pET 43.1 series includes three separate vectors (pET 43.1a-c(+)). Each encodes a different reading frame to facilitate insert cloning, a His·Tag® to ease purification, and a protease site to remove the tags if desired. These plasmids may be purchased separately for $131. They may also be obtained as part of the pET NusA Fusion System 43.1, which contains all three vectors, a CD containing sequence information, and host strain glycerol stocks. Researchers can purchase this system with ($373) or without ($310) competent cells.

--Amy Constantine (amyconstantine@hotmail.com)
1.R.G. Harrison, "Expression of soluble heterologous proteins via fusion with NusA protein," inNovations (Novagen), 11:4-7, June 2000.
For More Information
Novagen Inc.
(800) 526-7319
www.novagen.com

Advertisement
Keystone Symposia
Keystone Symposia

Follow The Scientist

icon-facebook icon-linkedin icon-twitter icon-vimeo icon-youtube
Advertisement
RayBiotech
RayBiotech

Stay Connected with The Scientist

  • icon-facebook The Scientist Magazine
  • icon-facebook The Scientist Careers
  • icon-facebook Neuroscience Research Techniques
  • icon-facebook Genetic Research Techniques
  • icon-facebook Cell Culture Techniques
  • icon-facebook Microbiology and Immunology
  • icon-facebook Cancer Research and Technology
  • icon-facebook Stem Cell and Regenerative Science
Advertisement
Advertisement
NeuroScientistNews
NeuroScientistNews