A novel system for expressing recombinant proteins over a wide temperature range from 4 to 35 degrees C.
Escherichia coli cells are the most commonly used host cells for large-scale production of recombinant proteins, but some proteins are difficult to express in E. coli. Therefore, we tested the nocardioform actinomycete Rhodococcus erythropolis, which grows at temperatures ranging from 4 to 35 degrees C, as an expression host cell. We constructed inducible expression vectors, where the expression of the target genes could be controlled with the antibiotic thiostrepton. Using these expression vectors, several milligrams of reporter proteins could be isolated from 1 liter of culture of R. erythropolis cells grown at a temperature range from 4 to 35 degrees C. Moreover, we successfully purified serum amyloid A1, NADH dehydorogenase 1 alpha subcomplex 4, cytochrome b5-like protein, apolipoprotein A-V, cathepsin D, pancreatic Rnase, and HMG-1 that are all difficult to express in E. coli. In the case of kallikrein 6, mouse deoxyribonuclease I and Kid1, which are also difficult to express in E. coli, the expression level of each protein increased when proteins were expressed at low temperature (4 degrees C). Based on these results, we conclude that a recombinant protein expression system using R. erythropolis as the host cell is superior to respective E. coli systems.
Nakashima
Nobutaka
N
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan.
Tamura
Tomohiro
T
D000626:Aminopeptidases / Q000096:biosynthesis / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012240:Rhodococcus / Q000235:genetics* / Q000378:metabolism*; D013045:Species Specificity; D013696:Temperature; D013883:Thiostrepton / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators; C089891:tipA protein, Streptomyces; D000626:Aminopeptidases; C022450:prolyl aminopeptidase; D013883:Thiostrepton