Various Expression System

Plant Expression System Baculovirus Expression System Yeast Expression System

Plant Expression Systems

High copy number plant vector, pNosdcGUS, expresses the GUS gene under the control of the nopaline synthase (pNos) promoter. This vector may be used to monitor virus production and transfection efficiency. Application: The GUS gene can be excised using the 5 XmaI and 3 SacI sites to allow the insertion of other genes to be expressed under the same regulatory elements in plants.

 The linked nucleic acid sequences further comprise multiple cloning sites. The nucleic acid segment encodes a plant virus movement protein. The nucleic acid segment encodes a plant virus coat protein. Nucleic acid sequences further comprise a marker gene or a selectable marker gene. Nucleic acid sequences further comprise nucleic acid which encodes a plant virus coat protein. Rather it can be said as nucleic acid sequences further comprise nucleic acid encoding a plant virus movement protein. The linked nucleic acid sequences encode a fusion polypeptide comprising the marker or selectable marker and the plant virus coat protein or the plant virus movement protein.

A nucleic acid composition, comprising: (a) isolated RNA-1 of flock house virus; and (b) a recombinant RNA molecule comprising linked RNA sequences comprising: RNA-1 or RNA2 of flock house virus and RNA encoding a plant virus coat protein. A method of expressing a gene product encoded by a recombinant RNA in a plant host cell, comprising: a. contacting the plant host cell with an amount of the composition of isolated RNA-1 of flock house virus, a recombinant RNA comprising the vector. b. detecting or determining whether the gene product encoded by the recombinant RNA is expressed.

Baculovirus Expression System

Vectors pUCDM and pFBDM contain expression cassettes comprising a central multiplication module (M), promoters (polh, p10), multiple cloning sites (MCS1, MCS2), and terminators (SV40, HSVtk) flanked by unique PmeI and AvrII (boxed) endonuclease sites.

pUCDM also contains the inverted repeat for CreloxP site-specific recombination (LoxP) and a resistance marker for chloramphenicol. pFBDM contains transposon elements (Tn7L, Tn7R) and resistance markers for ampicillin and gentamycin.

Genes of interest are cloned into MCS1 or MCS2. (b) Derivatives of pUCDM or pFBDM containing multigene expression cassettes are assembled by excising the expression cassette containing two genes (Gene A, Gene B) using PmeI and AvrII digestion. Inserting the fragment generated into another cassette containing more genes (Gene C, Gene D) via the BstZ17I/SpeI or the NruI/SpeI sites (both pairs PmeI/AvrII compatible) present in the multiplication module (M).

The process is iterative. (c) MultiBac is adaptable to combinatorial applications of protein production. Example (1) shows multiple genes encoding for protein subunits cloned in pUCDM using the multiplication module and inserted into MultiBac bacmid by Cre-loxP site-specific recombination.

In the same reaction, genes cloned singly in pFBDM encoding for a series of truncation variants of an additional protein are inserted into the bacmid via Tn7 transposition. Example (2) shows different enzymes cloned singly in pUCDM for insertion at LoxP with the purpose of modifying the proteins already expressed from Tn7.

Yeast Expression System