Honing Your Cloning

Early attempts to design vehicles for the cloning of foreign DNA produced vectors that were too big, unstable, or unselectable. The tide turned in 1977 with the construction of pBR313, the direct ancestor of the well-known pBR322, which forms the basis of many vectors that are still used extensively today. 1 However, the cloning systems introduced in the last year or so seem to be about as related to pBR313 as Ferraris are to little red Radio Flyer wagons. Some of the new protein expression systems allow an easily trackable component to be attached to the protein of interest to facilitate purification or other analyses. Others involve a melange of expression and purification features in a package that allows rapid shuttling of a gene of interest from scenario to scenario. The first generation of Escherichia coli expression vectors produced an in-frame fusion of the gene of interest and lacZ. 2 ß-galactosidase assays could be used to evaluate how much fusion protein was produced, and anti ß-galactosidase antibodies helped enormously with purification. Next came vectors that allowed cleavage of the fusion protein with proteases such as blood coagulation factor Xa. These systems yield the protein of interest without extraneous material attached. A recent variation on this theme is the IMPACT system, which generates a self-cleaving fusion protein. IMPACT is offered by New England Biolabs of Beverly, Mass., which recently updated the IMPACT T7 system to the more versatile IMPACT CN and IMPACT TWIN systems. Other newcomers to this arena are the pHAT vectors from CLONTECH of Palo Alto, Calif., which use protease EK cleavage sites to release the protein of interest. Stratagene's Complete Control Inducible Mammalian Expression System Vector Versatility The Gateway ™ Cloning Technology from Life Technologies of Rockville, Md., allows protein expression, rapid subcloning, and functional analysis of a gene of interest. To begin using the Gateway System, it is first necessary to create an "Entry" Clone. A variety of options exist for creating appropriately framed fusions between the gene of interest and one of several "Entry Vectors." This cloning can be performed through conventional approaches or through the "BP reaction" that uses attB recombination to insert the gene of interest. Optimized N- or C-terminal fusions for both E. coli and eukaryotic cells are available. After creating the Entry Clone, Gateway Cloning Technology allows rapid subcloning into a host of "Destination Vectors"--with no restriction digests, fragment purifications, or ligations. The Entry Clone and Destination Vectors contain the recombination sites attL, attR, attB, and attP from bacteriophage lambda. Mixing DNA from an Entry Clone with DNA from a Destination Vector, in the presence of a cocktail of purified enzymes, allows site-specific recombination between the two plasmids. The location of the attL, attR, attB, and attP sites in the vectors maintains the correct orientation of the cloned insert as it moves from the Entry to the Destination Vector; the precise positioning of the recombination sites maintains the correct reading frame for the protein fusions. 3 An LR Clonase ™ -mediated recombination reaction generates two products: an Expression Clone (the gene of interest in the Destination Vector) and an Entry Vector that now contains a segment from the original Destination Vector; this fragment contains the ccdB gene, which encodes a protein that prevents growth on most E. coli strains by interfering with DNA gyrase. This selection results in approximately 99 percent of transformants containing the correct insert. A selection of Destination Vectors allows native protein expression or fusions to polyhistidine or glutathione-S-transferase for expression in E. coli, insect cells, or mammalian cells (from T7, polyhedron, and SV40 promoters, respectively). Also, services and products are available to convert existing vectors into Destination Vectors. The Echo ™ system from Invitrogen Corp. of Carlsbad, Calif., and the Creator ™ system from CLONTECH are conceptually similar to the Gateway system, but they use Cre-loxP-mediated recombination from phage P1 to move fragments from one vector to another. 3 Echo uses single loxP sites on its donor and acceptor plasmids, and Cre-mediated recombination produces a fusion plasmid that contains the entire sequence of the donor and acceptor. Creator uses two loxP sites flanking its multiple cloning site, allowing solely the fragment of interest to be moved to the acceptor vector. Creator also incorporates the sacB gene between the loxP sites in the acceptor vector; this confers sensitivity to sucrose, allowing elimination of the donor material in the transformation. CLONTECH has modified several of its popular vector systems to be compatible with Creator, allowing a number of expression strategies and environments. C- terminal fusions to Living Colors ® fluorescent proteins permit fluorescent tracking during purification as well as studies on cellular localization of the product and FACS-based purification. Fusions to green, cyan, and yellow fluorescent proteins are available. Creator-compatible bicistronic expression vectors allow ready selection of transformed mammalian cells through cotranscription of the gene of interest with a neomycin resistance gene, while Creator-compatible Tet, RevTet, and retroviral expression systems give high levels of gene expression in insect and mammalian cell cultures. A ProTet Creator-compatible vector is designed for inducible gene expression in bacteria. Finally, two-hybrid analysis is also possible with Creator-compatible MATCHMAKER vectors. Similar to Gateway and Creator, Invitrogen's Echo system is compatible with several expression strategies (V5 and 6xHis fusions) and environments (promoters and vectors for expression in E. coli, yeast, insect, and mammalian cells). Echo also is compatible with Invitrogen's TOPO® cloning methods. TOPO cloning uses topoisomerase I from Vaccinia virus to insert target fragments into a vector in a novel way. Vaccinia topoisomerase I makes a sequence-specific cleavage in the DNA after 5' CCCTT and becomes covalently bound to the 3' end. By introducing opposing 5' CCCTT sequences into a plasmid, Invitrogen is able to purify a linear vector that has topoisomerase I covalently bound to both 3' ends. When provided with an appropriate substrate, the bound topoisomerase I will perform an efficient ligation. In this way, blunt-ended fragments (from PCR reactions performed with proofreading polymerases) can be cloned efficiently. A linear vector with a single 3' T overhang accepts fragments from nonproofreading- polymerase PCR, due to the tendency of nonproofreading enzymes to add a single, nontemplated, 5' A to the product. Novagen of Madison, Wis., has taken a different approach to addressing the common need for protein expression in several different environments. Its pTriEx ™ vector series permits optimum expression in E. coli, insect, and mammalian cells. 4 Vector pTriEx-4, for example, can use three different promoters to drive transcription of a gene of interest: an IPTG-inducible T7 promoter for E. coli, baculovirus p10 for insect cells, and a CMV promoter for mammalian cells. This vector can be transformed or transfected through standard methods, and genes can be cloned with two N-terminal and two C- terminal fusion tags. N-terminal options include S*Tag and His*Tag ® fusion sequences for homogenous assay and purification, respectively. In-frame (C-terminal) fusions can be made to an HSV*tag ® for detection and His*Tag. Thrombin and enterokinase protease cleavage sites permit target protein cleavage from fusion sequences during or after purification. Three other pTriEx vectors are currently available, including two for native protein expression. Two bicistronic vectors are due to be released this fall. Savvy Secretions In addition to the tags that are conventionally fused to proteins of interest, alkaline phosphatase--which is becoming a label of choice for many DNA applications--is being used in protein studies. GenHunter's AP-TAG ™, for example, is a vector that allows easy construction of in-frame fusions between a protein or peptide of interest and the gene for human-secreted alkaline phosphatase. When this fused gene is expressed in certain cell lines (293T cells) high levels of the protein are secreted into the culture medium. The system is designed to use these alkaline phosphatase-protein fusions as probes to study and purify cell-surface receptors and ligands, although the potential range of applications is much wider. The PhoA*Color ™ System from Qbiogene of Montreal is similar and is used to create alkaline phosphatase-tagged proteins from E. coli. The fusion used in this system relies on the PhoA signal peptide to export the protein of interest--fused to alkaline phosphatase--into the E. coli periplasmic space, which often means that relatively pure preparations can be obtained through simple purification protocols. The PhoA*Color system also uses the alkaline phosphatase gene for a colorimetric positive selection method during the cloning stage. When a protein of interest is fused to a tag such as GST, 6xHis, or a FLAG ® peptide from Sigma-Aldrich of St. Louis, the tag is often required to provide two somewhat exclusive functions: binding the protein to a substrate and quantifying how much protein is present. QIAGEN of Valencia, Calif., has created the DoubleTag System ™, part of the extensive QIAexpress Expression System, to help in this process. pQE-100 has a multiple cloning site located between an upstream sequence that encodes 6xHis and a downstream sequence that encodes a 12-amino acid Tag·100 epitope from mammalian MAP kinase 2. Expression of the fusion protein is driven by a lac operator- controlled phage T5 promoter. The N-terminally fused 6xHis tag allows purification of the fusion protein through nickel-based capture techniques (such as QIAGEN's Ni-NTA Magnetic Agarose Beads) and also allows the protein to be immobilized in a specific orientation using similar methods (for example, using QIAGEN's Ni-NTA HisSorb Plates). Once immobilized in this fashion, the Tag·100 epitope is still available to take part in other interactions. The pQE series of vectors does not incorporate proteolytic sites to allow separation of the protein of interest from the tag proteins (although introducing such sites during cloning is feasible). Even with the tags in place, however, proteins produced through this system have been used for a wide range of functional assays, antibody production, and even crystallographic studies. Expressionist Era Several new vectors for optimized or controlled expression are also becoming available. In the Complete Control ™ Inducible Mammalian Expression System from Stratagene of La Jolla, Calif., a synthetic ecdysone-inducible receptor and a synthetic receptor recognition element regulate transcriptional control in pEGSH and pERV. The artificial nature of this system ensures that endogenous control elements and factors in the host cells will not interfere with experiments. Inducible systems can also be used to overexpress toxic or disease-causing genes, induce gene targeting, and express antisense RNA. The vectors contain a multiple cloning site, tightly regulated promoters (CMV in pERV and a minimal SP1 in pEGSH), and selectable markers for both E. coli (kanamycin or ampicillin) and mammalian cells (G418 or hygromycin). Genes cloned in these vectors are transcriptionally inactive until the system is activated with the ecdysone analogs muristerone A or ponasterone A; over 1,000-fold activation has been observed in stable cell lines. The inducer exhibits no pleiotropic effects on cellular physiology, and the inducer's lipophilic nature and short in vivo half-life ensure that it rapidly penetrates and clears all tissues as well as exhibits dose-dependent control of gene expression. Stratagene has also introduced the ViraPort ™ retroviral gene expression system that uses the Moloney murine leukemia virus (MMLV)-based pFB and pFB-Neo vectors for constructing replication-incompetent virus particles. These particles serve as gene transfer vehicles to transduce virtually any cell line and isolate a particular gene of interest. The vectors have an enhanced version of the MMLV packaging signal, which leads to high viral titers, and also contain a splice acceptor sequence that results in high levels of protein expression from a cloned gene. An ampicillin resistance marker and bacterial origin of replication allow manipulations and maintenance in E. coli. Furthermore, all of the cis and trans elements required to produce virus are also removed. These are divided onto separate plasmids known as Stratagene's pVPack vectors, which include a gag-pol expressing gene along with a choice of four env-expressing genes to be selected depending on the types of cells the user wishes to transduce. Thus, a packaging cell line isn't necessary. By separating these viral coding sequences onto three separate plasmids, the probability of producing undesired replication-competent virus is very low. As such, pFB is capable of insertions of up to 8 kb (pFB-Neo allows approximately 7 kb) without significant loss of viral titer. Titers approaching 10 8 pfu/ml are normally achieved with this system. The ViraPort retroviral gene expression system offers distinct advantages when transfection efficiencies are low, copy-number control is advantageous, or cell types do not transfect well. Microbix Biosystems of Toronto also has addressed a common problem with mammalian expression systems: obtaining sufficient recombinant DNA to perform an experiment. Its AdMax system for adenovirus expression vector construction can result in up to 100-times better vector rescue than other two-plasmid systems. The gene of interest (or a cassette of genes, up to 8 kb) is cloned into a ~4 kb E. coli shuttle plasmid under the control of a mammalian promoter. This plasmid can be rapidly grown and purified in high yield (100 µg). The genomic plasmids in this system are around 34 kb and contain all the necessary components for transfection and expression in appropriate cell lines. The shuttle plasmid and genomic plasmids both contain either Cre recombinase and loxP recombination sites or Flp recombinase and FRT recombination sites. Cotransfection with compatible shuttle and genomic plasmids results in efficient formation of a hybrid molecule. Site-specific recombination systems are clearly beginning to play a major role in subcloning, eliminating many of the problems caused by unfortunate restriction site placement. It is difficult to conceive of experiments that cannot be addressed using currently available technology. But undoubtedly by the time LabConsumer revisits this topic there will be a host of jaw-dropping improvements. Stay tuned. Bob Sinclair (bobsinclair@tech-write-edit.com) is a freelance writer in Salt Lake City. References 1. F. Bolivar et al., "Construction and characterization of new cloning vehicles. II. A multipurpose cloning system," Gene, 2:95, 1977. 2. U. Ruther, B. Muller-Hill, "Easy identification of cDNA clones," EMBO Journal, 2:1791-4, 1983. 3. A. Francis, "Pain-free cloning?" The Scientist, 14[2]:21, Jan. 24, 2000. 4. A. Constans, "Have it three ways," The Scientist, 14[7]:24, April 3, 2000.

Honing Your Cloning

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Bob Sinclair

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August 2000

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