Despite exciting progress in deciphering and decoding the genome, expression of gene products into functional proteins, especially in high-throughput applications, remains a complex challenge. Researchers are tackling those issues head-on by developing new tools and strategies.
CHI’s upcoming “Engineering Genes, Vectors, Constructs and Clones” conference will highlight some of these emerging approaches including rapid parallel cloning, redesigning more efficient promoters, and novel technologies for metabolic and glycoengineering.
Protein expression requires two initial critical decisions: the selection of a cloning strategy (e.g., deciding what vector properties are needed such as type of promoter, addition of a fusion tag, etc.), and selection of the cell type for expression (i.e., prokaryotic versus eukaryotic). High-throughput protein expression adds other requirements: the necessity for automatable methods for rapid cloning and protein isolation.
Development of a ligation-independent method of cloning is a key element, says W. Clay Brown, Ph.D., scientific director of the high-throughput protein lab, Center for Structural Biology, Life Sciences Institute, University of Michigan.
“It used to be that both the gene and vector were digested with a compatible restriction endonuclease site and then ligated together. The problem is restriction sites may not be compatible across a number of genes and may need constant re-adjustment.
“Currently, newer technology has emerged, allowing us to perform ligation-independent cloning (LIC) that universalizes our ability to use any sequence. This has clear advantages for high-throughput gene expression where the whole idea is to do many variations at once rather than one clone at a time.”
Dr. Brown’s group is expressing proteins in insect cells utilizing a series of new LIC baculovirus vectors.
“We developed a series of plasmids for optimizing LIC cloning. These can assess the effect of different fusion proteins by themselves or in combination with different signal peptides. They can be utilized for expressing intracellular or secreted proteins. Use of a removable histidine tag allows rapid batch purification of expressed proteins.”
Baculovirus expression has several advantages. “Such systems allow high-level protein expression in insect cell suspension cultures. Importantly, because insect cells are eukaryotic, they also allow proper folding and inclusion of post-translational modifications such as glycosylation. Prokaryotic systems cannot do this.”
The combination of quick and efficient LIC cloning technologies with high-level eukaryotic expression is helping to usher protein expression into the high-throughput arena.
“Developing target proteins that are relevant to human diseases requires a lot of optimization to obtain a protein with the necessary therapeutic properties. Once you have obtained this, the hope is to be able to ramp up the system to produce high levels of product for subsequent therapeutic development or structural studies.”