RESUMO
The rate-limiting step in protein production is usually the generation of an expression clone that is capable of producing the protein of interest in soluble form at high levels. Although cloning of genes for protein expression has been possible for some time, efficient generation of functional expression clones, particularly for human proteins, remains a serious bottleneck. Often, such proteins are hard to produce in heterologous systems because they fail to express, are expressed as insoluble aggregates, or cannot be purified by standard methods. In many cases, researchers are forced to return to the cloning stages to make a new construct with a different purification tag, or perhaps to express the protein in a different host altogether. This usually requires identifying new cloning schemes to move a gene from one vector to another, and frequently requires multistep, inefficient cloning processes, as well as lengthy verification and sequence analysis. Thus, most researchers view this as a linear pathway - make an expression clone, try it out, and if it fails, go back to the beginning and start over. Because of this, protein expression pipelines can be extremely expensive and time consuming.The advent of recombinational cloning has dramatically changed the way protein expression can be handled. Rapid production of parallel expression clones is now possible at relatively low cost, opening up many possibilities for both low- and high-throughput protein expression, and increasing the flexibility of expression systems that researchers have available to them. While many different recombinational cloning systems are available, the one with the highest level of flexibility remains the Gateway system. Gateway cloning is rapid, robust, and highly amenable to high-throughput parallel generation of expression clones for protein production.
Assuntos
Clonagem Molecular/métodos , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Animais , Baculoviridae/genética , Escherichia coli/genética , Expressão Gênica , Oligonucleotídeos/química , Reação em Cadeia da Polimerase/métodos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes/química , Recombinação Genética/genética , Leveduras/genéticaRESUMO
Lentiviral expression clones, which contain long direct repeats, often show dramatic instability in Escherichia coli, leading to difficulties in obtaining valid clones. We show that the reduced-genome E. coli strain MDS42 is capable of stabilizing lentiviral expression clones containing direct repeats, and outperforms many commonly used cloning strains for this purpose. In addition, the strain has several characteristics that make it highly amenable for use in recombinational cloning systems.
