Rapid subcloning using site-specific recombination
a site-specific recombination and subcloning technology, applied in the direction of stable introduction of dna, biochemistry apparatus and processes, microorganisms, etc., can solve the problems of time-consuming purification of the subcloning intermediate, and the inability to transfer the desired coding region to an expression vector
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example 1
Construction of Univector Constructs
[0162] In this example, illustrative Univector constructs are provided. The map for several Univectors is shown in FIG. 23, showing pUNI-10, pUNI-20, and pUNI-30. In this figure, nucleotide positions (in parentheses) of unique restriction enzyme cleavage sites are shown. Functional sequences are shown as filled boxes and are labeled inside of the circle. Boxes with arrows are genes transcribed in the direction of the arrow. Below each map is the sequence of the polylinker region displayed as coding triplets in frame with the open reading frame of loxP. Unique restriction enzyme cleavage sites are in bold. General features of these Univectors include a loxP site placed adjacent to the 5′ end of a polylinker for insertion of cDNAs. loxP has a single open reading frame that is in frame with the ATG of the NdeI and NcoI sites of the polylinker. This facilitates the subsequent generation of protein fusions as noted below. Following the polylinker are ...
example 2
Construction of Host Plasmids for Use in the Univector Plasmid-Fusion System
[0167] Host plasmids used in the Univector plasmid fusion system are referred to as pHOST plasmids pHOST plasmids or vectors are generally expression vectors that have been modified by the insertion of a site-specific recombination site, such as a lox site. The presence of the lox site on the pHOST plasmid permits the rapid subcloning or insertion of the gene interest contained within a pUNI vector to generate an expression vector capable of expressing the gene of interest The pHOST vector may encode a protein domain such as an affinity domain including, but not limited to, glutathione-S-transferase (Gst), maltose binding protein (MBP), a portion of staphylococcal protein A (SPA), a polyhistidine tract, etc. A variety of commercially available expression vectors encoding such affinity domains are known to the art. When the pHOST plasmid contains a vector-encoded affinity domain, a fusion protein comprising ...
example 3
Expression and Purification of a Gst-Cre Fusion Protein
[0181] In order to provide a source of purified Cre recombinase for the in vitro recombination of plasmids, the cre gene was inserted into a Gst expression vector such that a fusion protein comprising Gst at the amino-terminal end, and Cre recombinase at the carboxy-terminal end was produced. The Gst-Cre fusion protein was purified by chromatography using Glutathione Sepharose 4B (Pharmacia). Purified Gst-Cre can be stored at −80° C., −20° C., or 4° C. for several months without significant loss of activity.
[0182] To simplify Cre purification, a plasmid expressing a GST-cre fusion protein was constructed, pQL123. The cre gene was isolated by polymerase chain reaction (PCR) amplification using the plasmid pBS39 (U.S. Pat. No. 4,959,317). U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,965,188 describe PCR methodology and are incorporated herein by reference. The primers used in the PCR were designed to introduce an NcoI site at the f...
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