SAMY, a novel mammalian reporter gene derived from Bacillus stearothermophilus alpha-amylase.
The Bacillus stearothermophilus alpha-amylase (amyS) is a heat-stable monomeric exoenzyme which catalyses random hydrolysis of 1,4-alpha-glucosidic linkages in polyglucosans. The Bacillus alpha-amylase was engineered for use as an intracellular (AmyS(Delta S)) as well as a secreted reporter protein (SAMY; secreted alpha-amylase) in mammalian cells. The 5' end of amyS containing the prokaryotic secretion signal was either deleted (amyS(Delta S)) or replaced by a murine immunoglobulin secretion signal. SAMY was cloned under control of the cytomegalovirus promoter (P(CMV)) in a mammalian expression vector or the promoter of the human elongation factor 1 alpha (P(EF1 alpha)) in a lentiviral expression context. A variety of mammalian and human cell lines growing as monolayers, in suspension or as three-dimensional spheroids were transfected/transduced with SAMY- or amyS(Delta S)-encoding expression/lentiviral vectors and alpha-amylase activity was measured in cell lysates and culture supernatants. These experiments showed that SAMY and AmyS(Delta S) were either secreted or remained intracellular as highly sensitive reporter enzymes. SAMY expression and detection was fully compatible with established SEAP (human secreted alkaline phosphatase) and u-PA(LMW) (low molecular weight urokinase-type plasminogen activator) reporter systems and could be used to quantify expression of up to three independent genes in one culture supernatant.
Schlatter
Stefan
S
Institute of Biotechnology, Swiss Federal Institute of Technology, ETH Hoenggerberg, CH-8093 Zurich, Switzerland.
Rimann
Markus
M
Kelm
Jens
J
Fussenegger
Martin
M
D016475:3T3 Cells; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D003124:Colorimetry; D006224:Cricetinae; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D020014:K562 Cells; D051379:Mice; D014162:Transfection; D014407:Tumor Cells, Cultured; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism
D000516:alpha-Amylases