From no expression to high-level soluble expression in Escherichia coli by screening a library of the target proteins with randomized N-termini.
For structural studies by x-ray crystallography and nuclear magnetic resonance it is important for the target protein to be available in large quantity and high purity. Escherichia coli expression systems remain the most versatile and convenient means to produce a large quantity of recombinant proteins. Unfortunately, some proteins fail to be expressed in E. coli or are expressed in an insoluble form. To overcome the difficulty of no expression or expression at a very low level, a simple and efficient approach of screening a library of variants of a target protein with randomized N-termini was devised. In this method, a few N-terminal residues are randomized by designing a mixture of oligonucleotides for the forward PCR primer and we fuse the library in front of green fluorescent protein, which serves as a reporter for the target protein expression level and folding yield. In favorable cases this approach can result in high-level soluble expression of recombinant proteins in E. coli. This chapter describes the results of a test of this approach with a bacterial protein (the HI0952 gene product) that is not well expressed in E. coli.
Kim
Kyoung Hoon
KH
Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul, Korea.
Yang
Jin Kuk
JK
Waldo
Geoffrey S
GS
Terwilliger
Thomas C
TC
Suh
Se Won
SW
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D015723:Gene Library; D049452:Green Fluorescent Proteins / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins