Cell-free synthesis and in situ isolation of recombinant proteins.
We present a method for rapid expression and isolation of recombinant proteins. Cell-free protein synthesis in the presence of affinity beads enables in situ isolation of translation products, which simplifies the procedures for the preparation of purified protein samples. In the present study, we have made an attempt to carry out in situ isolation of histidine-tagged proteins by using Ni-NTA magnetic agarose beads. The presence of Ni-NTA beads gave no drastic effects on the efficiency of protein synthesis and successfully captured the synthesized proteins. Purified proteins were obtained after subsequent washing and elution steps. In particular, most of the endogenous bead-binding proteins were removed by pre-treating S30 extract with affinity beads and the purity of the target proteins was enhanced up to 95%. The methods described here will provide a basis for fast and convenient preparation of purified proteins from multiple genetic sequences.
Kim
Tae-Wan
TW
School of Chemical and Biological Engineering, College of Engineering, Seoul National University, Seoul 151-742, Republic of Korea.
Oh
In-Seok
IS
Ahn
Jin-Ho
JH
Choi
Cha-Yong
CY
Kim
Dong-Myung
DM
D002474:Cell-Free System; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008280:Magnetics; D009532:Nickel / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D012685:Sepharose / Q000737:chemistry
D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D009532:Nickel; D012685:Sepharose