Recombinant Protein Expression - High-Throughput Gene-Cloning Systems

Protein expression is a process in which proteins are synthesized, modified and regulated. Protein expression involves laboratory techniques & procedures which aid in manufacture of proteins.

The technique allows to produce and purify the desired protein, either inside or outside a cell. The proteins synthesized using protein expression technique can be used for industrial processes or to diagnose and to treat diseases. Protein expression provides substrates or enzymes required for further analysis.

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After obtaining target genes, the next step is high-throughput construction of expression vectors. Various cloning methods have been developed to make the process simple, time-efficient and cost-effective. Based on the underlying principle, the methods can be classified as restriction enzyme (RE)-based cloning, recombination-based cloning, and annealing-based or ligation-independent cloning (LIC).

The advantages and limitations of these methods have been discussed in previous reviews. In recent years, vast improvements in these methods have been made. Here, we concentrate on the most basic principles and the latest innovations in the existing methods.

RE-based cloning performed with DNA ligation has been used for four decades, but it was previously considered to be unsuitable for high-throughput methods because appropriate and compatible REs must be selected for each cloning procedure. The method has received increased attention since 2006, when SgfI and PmeI, the two most rare-cutting REs in the human DNA, were used and the Flexi Cloning system was developed by Promega (Madison, WI, USA).

The combination of SgfI and PmeI has been suggested to allow the cloning of more than 95% of genes of selected model organisms. The experimental procedure is similar to that use for conventional RE-based cloning: target genes are amplified using primers containing adapter sequences and then digested by two enzymes.

The vector is also digested, releasing highly toxic barnase gene for lethal selection, which can be used as a marker against the parental vector. Subsequently, the target gene and vector are ligated and transformed into competent cells. Nagase et al. used the Flexi Cloning system to produce proteins from 1929 open reading frame clones of human genes, demonstrating that this system can be successfully used in a high-throughput manner.

source: royalsocietypublishing.org