Ligation independent cloning vectors for expression of SUMO fusions.
With demand increasing for the production of many different proteins for biophysical or biochemical analyses, rapid methods are needed for the cloning, expression and purification of native recombinant proteins. In particular, generic methods are required that are independent of the target gene sequence. To address this challenge we have constructed four Escherichia coli expression vectors that can be used for ligation independent cloning (LIC) of an amplified target gene sequence. These vectors represent the combinatorial pairing of two different parent vector backbones with two different affinity tags. The target gene is cloned downstream of the sequence coding for an affinity-tagged small ubiquitin related modifier (SUMO). Using enhanced green fluorescent protein (eGFP) as an example we demonstrate that the LIC procedure works with high efficiency for all four of the vectors. We also show that the resultant recombinant SUMO fusion proteins can be overexpressed in E. coli and readily isolated by standard affinity purification techniques. Importantly, the purified fusion product can be treated with recombinant SUMO hydrolase to yield a mature target protein with any residue except proline at the amino terminus. We demonstrate an application of this by generating recombinant eGFP containing a non-native amino terminal cysteine residue and using it as a substrate for expressed protein ligation (EPL). The reagents and techniques described here represent a generic method for the rapid cloning and production of a target protein, and would be appropriate for a high throughput genomic scale expression project.
Weeks
Stephen D
SD
Department of Biochemistry & Molecular Biology, Drexel University College of Medicine, 245 N 15th Street, Mailstop 497, Philadelphia, PA 19102-1192, USA.
Drinker
Mark
M
Loll
Patrick J
PJ
D000067699:Ataxin-3; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004271:DNA, Fungal; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D005822:Genetic Vectors; D016681:Genome, Fungal; D018698:Glutamic Acid / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D008156:Luciferases / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques; D010957:Plasmids; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D004271:DNA, Fungal; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D049452:Green Fluorescent Proteins; D018698:Glutamic Acid; D006639:Histidine; D008156:Luciferases; C092341:ATXN3 protein, human; D000067699:Ataxin-3; D003546:Cysteine Endopeptidases; C118083:Ulp1 protease