Application of the wheat-germ cell-free translation system to produce high temperature requirement A3 (HtrA3) proteases.
Mammalian high temperature requirement A3 (HtrA3) is a serine protease of the HtrA family. It is an important factor for placental development and a tumor suppressor. The biochemical properties of HtrA3 are uncharacterized. One critical step in biochemical characterization is overexpressing and purifying the full-length recombinant protein. However, utility of cell-based expression systems is limited for a protease because of autocleavage. The wheat-germ cell-free translation system is highly efficient at producing "difficult" eukaryotic multidomain proteins and is easily modifiable for protein synthesis at different temperatures. In this study, we evaluated the potential of the wheat-germ cell-free translation system for producing human HtrA3. HtrA3 underwent autocleavage when synthesized at 17 °C. When the synthesis temperature was lowered to 4 °C, full-length HtrA3 was successfully produced and proteolytically active. Catalytic site serine substitution with alanine (S305A) stabilized HtrA3 while abolishing its protease activity. This mutant was readily synthesized and stable at 17 °C. When used with glutathione S-transferase (GST) pull-down assay, S305A HtrA3 was a valuable bait in searching for endogenous HtrA3 binding proteins. Thus, we demonstrated the unique utility of the wheat-germ cell-free translation system for producing and characterizing human HtrA3. These strategies will be likely applicable to a wide range of proteases.
Prince Henry's Institute of Medical Research, Clayton, Victoria, Australia.
Harmeet
H
Singh
Shin-ichi
S
Makino
Yaeta
Y
Endo
Ying
Y
Li
Andrew N
AN
Stephens
Guiying
G
Nie
D019943:Amino Acid Substitution; D002474:Cell-Free System / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D009218:Myosins / Q000276:immunology / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D011506:Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D013696:Temperature; D014908:Triticum
D011506:Proteins; C474013:HTRA3 protein, human; D012697:Serine Endopeptidases; D009218:Myosins