Restriction sites for pgex4t1 the following restriction site table was compiled using dnasis software for sequence analysis. Ge healthcare gst gene fusion system pgex vectors 4t1 ge healthcare gst gene. The expanded mcs facilitates the unidirectional cloning of cdna inserts obtained from libraries constructed using many available lambda vectors. The pgex vectors have an expanded multiple cloning site mcs that contains six restriction sites. Expression, purification and anticancer analysis of gst.
For convenience, use the pgex 5 and 3 sequencing primers see appendix 4, sequencing of pgex fusions for more information on the. The codons within the nterminus of the vp1 protein were optimized using prediction software and changed to the preferred codon usage for e. Initial purification screen within a structural genomics program, amersham biosciences, code number 18115020. The li recombinant protein expression system has been, and still is. This saves you time and headaches compared to receiving synthetic gene constructs in a cloning vector and then having to do tedious cloning in your own lab. Data file 28962284 aa gst gene fusion system glutathione stransferase gst. Initial purification screen within a structural genomics program, amersham biosciences, code number 18 115020.
Genscripts express cloning service makes it fast, inexpensive, and easy to get readytouse genes cloned into the expression vector of your choice. Bacterial vector for expressing fusion proteins with a thrombin site. Troubleshooting in pgex expression vectors sigmaaldrich. Multiple sequence alignment of the putative amino acid sequence of xylanase with other related xylanases was carried out using the clustalw software. Construction and expression of prokaryotic expression. Expression vectors fused with genes of magnaporthe oryzae 10831 isolated from the digestion products using gel recovery and purification. This page describes the characteristics of pgex expression vectors used with ge. Sticky ends from different ahdi sites may not be compatible. Sticky ends from different bsai sites may not be compatible. To construct the expression vectors for prokaryotic and eukaryotic human augmenter of liver regeneration halr and to study their biological activity. I have to prepare a presentation about this topic for my biochemistry laboratory and i.
Why we use it and what are those pgex 2k and stuff. Not recommended or intended for diagnosis of disease in. Carl novinas lab contains the insert argonaute 2 and is published in mol cell. This saves you time and headaches compared to receiving. A novel cloning approach allowed us to generate a stable clone in the prokaryotic. The enzymes chosen are those which we believe to have been commercially. Terms and conditions 20042018 harvard medical school plasmid was created and is maintained by the dfhcc dna resource core at harvard medical schooldfhcc dna resource core at harvard. Dilute an overnight culture transformed with pgex fusion construct, 1. Furthermore, xynb geneamplified product was subcloned into pgex4t1 expression vector designated as pgex4t1xynb and was expressed in e. Rapid purification of gst fusion proteins using gstrap ff 1 ml and 5 ml columns. Phylogenetic analysis and prokaryotic expression of nmaap1. The pgex4t1 vector is a bacterial vector for expressing gst fusion proteins with a thrombin site. The expanded mcs facilitates the unidirectional cloning of cdna inserts obtained from libraries. This is a free resource for the scientific community that is compiled.
The pcr product of perforin was restricted with eco r i and not i restriction enzymes and ligated using t4. A putative rt gene was obtained by pcr and was ligated into various prokaryotic expression vectors. Vector database is a digital collection of vector backbones assembled from publications and commercially. It appears that my gene is not ligated to the vector.
The imagej software was used to compare fusion protein expression. A repository of over 200,000 plasmids including protein structure initiative protein expression plasmids and vectors, over 75,000 human plasmids, and whole genome collections from many organisms. Cloning the gene or gene fragment into a pgex expression vector. Data file 28962284 aa gst gene fusion system glutathione. After 35 cycles of amplification at 95c for 1 min, at 64c for 1min, at 72c for. The lac repressor binds to the lac operator to inhibit transcription in e. Expression, purification and bioactivity of human augmenter of liver. A strategy for highlevel expression of soluble and functional human. This vector contains the recognition sequence for the catalytic subunit of campdependent protein. The amplified product 35 bp was cloned into the plasmid pgex4t1 invitrogens. To complement the pgex vectors, gst vector primers for sequencing are available for immediate use in sequencing doublestranded dna inserted into the pgex vectors.
This is a free resource for the scientific community that is compiled by addgene. Vector database is a digital collection of vector backbones assembled from publications and commercially available sources. As part of that effort, we supply carefully annotated files for common plasmids. T, pgex6p1, pgex4t1, and pgex5x1 can directly accept and express cdna inserts isolated from. An expression vector has features that any vector may have, such as an origin of replication, a selectable marker, and a suitable site for the insertion of a gene such as the multiple. To see this sequence with restriction sites, features, and translations, please download snapgene or the free snapgene viewer. Cloning junctions should be sequenced to verify that insert is inframe.
Shop online for a wide selection of ge healthcare gst gene fusion system pgex vectors designed for inducible. Collectively, the pgex vectors provide all three translational reading frames beginning with the ecori restriction site. The prokaryotic expression vector pgex4t1 was simultaneously. Ge healthcare gst gene fusion system pgex vectors 4t1 ge. Simplified package with only the pgex vector in the pack e. To construct the expression vectors for prokaryotic and eukaryotic.
The recombinant vector and pgex4t2halr were identified by enzyme. Duplication and transcriptional divergence of three kunitz. T, pgex 6p 1, pgex 4t 1, and pgex 5x 1 can directly accept and express cdna inserts isolated from. Ge healthcare pgex4t1 gst expression vector product specification sheet code. Ben neels lab contains the insert shp2 and is published in mol cell biol 2000 jan. Find the right expression vector for your orf clone from our comprehensive list of vectors. T, pgex6p1, pgex4t1, and pgex5x1 can directly accept and express. The pgex4t1 vector was selected as the expression vector. Not recommended or intended for diagnosis of disease in humans or animals. The 1base overhangs produced by ahdi may be hard to ligate.
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