Gateway Cloning for Protein Expression
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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.
- Hartley, J. L., Temple, G. F., and Brasch, M. A. (2000) DNA cloning using in vitro site-specific recombination. Genome Res. 10, 1788–1795. CrossRef
- Kapust, R. B., Tozser, J., Fox, J. D., Anderson, D. E., Cherry, S., Copeland, T. D., and Waugh, D. S. (2001) Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutatnts with wild-type catalytic efficiency. Protein Eng. 14, 993–1000. CrossRef
- Studier, F. W. (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234. CrossRef
- Shine, J. and Dalgarno, L. (1975) Determinant of cistron specificity in bacterial ribos-omes. Nature 254, 34–38. CrossRef
- Horton, R. M., Cai, Z. L., Ho, S. N., and Pease, L. R. (1990) Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. Biotechniques 8, 528–535.
- Luckow, V. A., Lee, S. C., Barry, G. F., and Olins, P. O. (1993) Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. J. Virol. 67, 4566–4579.
- Kapust, R. B., Tozser, J., Copeland, T. D., and Waugh, D. S. (2002) The P1′ specificity of tobacco etch virus protease. Biochem. Biophy. Res. Commun. 294, 949–955. CrossRef
- Esposito, D., Gillette, W. K., and Hartley, J. L. (2003) Blocking oligonucleotides improve sequencing through inverted repeats. Biotechniques 35, 914–920.
- Hammarstrom, M., Hellgren, N., van den Berg, S., Berglund, H., and Hard, T. (2002) Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli. Protein Sci. 11, 313–321. CrossRef
- Gateway Cloning for Protein Expression
- Book Title
- High Throughput Protein Expression and Purification
- Book Subtitle
- Methods and Protocols
- pp 31-54
- Print ISBN
- Online ISBN
- Series Title
- Methods in Molecular Biology
- Series Volume
- Series ISSN
- Humana Press
- Copyright Holder
- Humana Press
- Additional Links
- Recombinational cloning
- Protein expression
- HTP cloning
- Site-specific recombination
- att sites
- Fusion proteins
- Solubility tags
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