Promoter-cDNA-directed heterologous protein expression in Xenopus laevis oocytes.
Heterologous proteins can be expressed in Xenopus laevis oocytes by cytoplasmic microinjection of mRNA. To circumvent limitations inherent in this approach we investigate direct nuclear injection of strong viral expression vectors to drive transcription and subsequent translation of cDNAs encoding cytoplasmic, secreted, and plasma membrane proteins. After several viral promoters had been tested, the pMT2 vector was found to be a superior expression vector for X. laevis oocytes capable of directing expression of high levels of functional heterologous proteins. Typically the amount of protein derived from transcription-translation of the microinjected cDNA accounts for approximately 1% of total non-yolk protein. Moreover, the inefficiency usually associated with nuclear injections was overcome by coinjection of pMT2 driving expression of a secreted alkaline phosphatase as an internal control to select positive-expressing oocytes. Using this method, we have successfully expressed high levels of chloramphenicol acetyltransferase, the adipocyte-specific cytosolic 422(aP2) protein, and the membrane-associated glucose transporter GLUT1. The system described should be applicable to a wide variety of proteins for which cDNAs are available. Hence, the cumbersome and often inefficient in vitro synthesis of mRNA for studying ion channels, receptors, and transporters as well as for expression cloning in Xenopus oocytes should no longer be necessary.
Swick
A G
AG
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Janicot
M
M
Cheneval-Kastelic
T
T
McLenithan
J C
JC
Lane
M D
MD
D000469:Alkaline Phosphatase / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006601:Hexoses / Q000378:metabolism; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D009865:Oocytes; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism*; D014982:Xenopus laevis / Q000235:genetics*
D006601:Hexoses; D009004:Monosaccharide Transport Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D000469:Alkaline Phosphatase