The KickStart Protein Expression Kit offers high-level, inducible expression of recombinant proteins in S. cerevisiae and a novel tag for easy purification of expressed proteins. KickStart vectors feature G418 selection and are propagated in rich medium to significantly enhance cell densities and protein yield. KickStart kits include the pKS-1-ST Strep-tag® expression vector, a second vector with no purification tag, and the pKS1-ST-Gal control vector. Available separately is the pKS2-ST Strep-tag® secretion vector.
Protein expression, under control of the ADH2 promoter, is induced when glucose in the growth medium is depleted, thus eliminating the need to add reagents or change the media to induce expression. Recombinant proteins are expressed in the S. cerevisiae strain DSY-5, which lacks the two major vacuolar proteases responsible for protein degradation, pep4 and prb1. Extracts prepared from DSY-5 show significantly less protein degradation than extracts from wild-type strains.
Optimized extraction buffers and glass beads for cell lysis are provided with the kit. Purification of tagged recombinant protein from the lysate is based on the well-known binding of biotin to streptavidin. The novel Strep-tag®/Strep-Tactin® system developed by IBA GmbH* uses an 8 amino acid peptide capable of binding to the biotin binding pocket of streptavidin. Engineered streptavidin (Strep-Tactin) is the perfect partner for this novel tag; the combination offers rapid, one-step purification under physiological conditions for proteins of unsurpassed purity. Strep-Tactin purification products can be purchased separately in the US from either Novagen or QIAGEN.
Kit Contents: pKS1 expression vector (5 ug), pKS1-ST expression vector (5 ug), pKS1-ST-Gal control vector (5 ug), DSY-5 yeast host strain, XT extraction buffer (50 ml), and Glass Beads (20 ml).
*Strep-tag® technology for protein purification and detection is covered by US patent 5506121, UK patent 2272698 and French patent 9313066. Purchase of reagents related to these technologies provides a license for non-profit and in-house research use only. Expression or purification or other applications of above-mentioned technologies for commercial use require a separate license from IBA GmbH (www.iba-go.com/contact.html).
pKS2-ST Strep-tag Secretion Vector (Cat # P03308)
Optional vector for use with the KickStart Protein Expression Kit (#P01006). KickStart expression vector with inducible ADH2 promoter, followed by a SUC2 (invertase) derived secretion signal, a Strep-tag for protein purification, an HA epitope tag for protein detection and a multiple cloning site. The vector backbone contains the KanMX cassette for selection in rich medium. Carries ampicillin resistance for selection in E. coli.