Protein purification via temperature-dependent, intein-mediated cleavage from an immobilized metal affinity resin.
The intein that interrupts the DNA polymerase II DP2 subunit in Pyrococcus abyssi can be overexpressed in Escherichia coli and purified as an unspliced precursor. On in vitro incubation at 37 degrees Celsius or higher, the intein mediates efficient protein splicing. Mutations can be introduced into an intein fusion protein that prevent the second and third steps of protein splicing. As a result, the intein fusion protein can facilitate temperature-dependent formation of a thioester linkage between the N-extein and intein. This thioester is susceptible to in vitro hydrolysis or thiolysis at temperatures of 40 degrees Celsius or higher, and we have exploited this activity to generate a temperature-dependent protein purification scheme. Protein purification using this intein does not require the addition of exogenous thiols and is compatible with the use of immobilized metal affinity chromatography. The identity of the C-terminal residue of the N-extein has less influence on the cleavage reaction than in current purification systems in terms of premature in vivo cleavage and is complementary to current systems in terms of efficient in vitro cleavage.
Mills
Kenneth V
KV
Department of Chemistry, College of the Holy Cross, Worcester, MA 01610, USA. kmills@holycross.edu
Connor
Katherine R
KR
Dorval
Deirdre M
DM
Lewandowski
Katherine T
KT
D000345:Affinity Labels; D001483:Base Sequence; D004257:DNA Polymerase II / Q000235:genetics / Q000302:isolation & purification; D019641:DNA, Archaeal / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D066298:In Vitro Techniques; D047668:Inteins; D010957:Plasmids / Q000235:genetics; D019154:Protein Splicing; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification*; D043903:Pyrococcus abyssi / Q000201:enzymology / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D012117:Resins, Synthetic; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
D000345:Affinity Labels; D019641:DNA, Archaeal; D011506:Proteins; D011993:Recombinant Fusion Proteins; D012117:Resins, Synthetic; D004257:DNA Polymerase II