January 1, 2012 (Vol. 32, No. 1)
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.”
Although baculovirus expression systems can produce glycosylated proteins, they cannot glycosylate the products identically to mammalian cells because insect cells have more primitive glycosylation pathways. Thus, insect cells are unable to produce recombinant glycoproteins containing the terminally sialylated complex-type N-glycans found on many mammalian glycoproteins.
“This is a limitation of baculovirus expression systems because those N-glycans have essential roles in glycoprotein function,” notes Donald Jarvis, Ph.D., professor, molecular biology, University of Wyoming.
“The production of recombinant therapeutic glycoproteins requires an expression system that can provide appropriate glycosylation to ensure what is produced is functionally and structurally similar to the native product. Since these systems cannot produce sialylated glycoproteins, this is a significant problem.”
Dr. Jarvis says that this problem can be addressed by utilizing glycoengineering approaches. “Over the past 15 years, we’ve clearly demonstrated that one can transform insect cells with genes encoding the N-glycan processing functions needed to ‘humanize’ their protein glycosylation pathways.
“The basic idea is to reprogram the endogenous insect cell glycosylation pathways, which enables insect cells to produce recombinant glycoproteins with human-type carbohydrate side chains.”
However, the system is not yet ready for prime time. “The current challenge is to produce transgenic insect cell lines that provide higher efficiencies of human-type glycan processing. Although we can successfully glycoengineer insect cells to humanize their glycosylation pathways, humanized N-glycan processing efficiencies have not been high enough to provide the level of recombinant glycoprotein homogeneity we’d like.
“Of course, this is a high standard because even the most widely used mammalian expression systems, such as Chinese hamster ovary cells, produce structurally heterogeneous recombinant glycoproteins,” he continues.
“We’re not sure when we’ll get there, but we intend to engineer the baculovirus-insect cell system to achieve this higher standard. This will greatly facilitate the production of better, more ‘mammalianized’ recombinant proteins for research and therapeutic applications.”
Similar to higher eukaryotes, the yeast Pichia pastoris also possesses biological machinery for engineering post-translational modifications of expressed proteins. They add another advantage: high-level expression of functional protein.
“Pichia provides an ideal solution for production of therapeutic or industrial proteins particularly because of its inexpensive growth medium, a high capability for secreting proteins, and scalability,” says Roland Weis, Ph.D., head of operations, VTU Technology.
Although not usually considered a high-throughput-amenable system, Dr. Weis says VTU has developed technology to bring it into that arena.
“Pichia possesses one of the strongest eukaryotic promoters known. It controls an alcohol oxidase (AOX) gene. While Pichia grows on methanol, it is repressed on glucose, glycerol, or ethanol. This provides a capability for tight regulation. We developed a microplate-based system for cultivation and subsequent screening of about 20,000 clones per week.
“The basis for this technology is our library of AOX1-promoter variants that allow for methanol-free as well as methanol-dependent protein expression. Because there are subtle differences in the expression by each promoter, we perform optimization of protein expression on a microscale for high-throughput screening in unrivaled timelines,” he explains.
“Our goal is to get the best expression for each construct. Screening of promoter variants is the way to accomplish that. There really is no particular rationale for why one promoter version works better than another. Nature finds its way differently. We let nature decide and then choose what nature chooses.
“Thus, high-throughput screening of our promoter library with any gene of interest allows us to optimize yeast expression of specific clones. After we find the ideal match, we can then directly scale up in a fermenter to produce high levels of that protein. In this way we are able to maximize throughput as well as output,” Dr. Weis concludes.
Another extensively utilized yeast expression system is that of Saccharomyces cerevisiae. It has become a platform organism in the field of metabolic engineering, which seeks to improve cellular activities and properties (e.g., the production of a metabolite) by genetic manipulation.
Nancy DaSilva, Ph.D., professor, chemical engineering and materials science, biomedical engineering, and Suzanne Sandmeyer, Ph.D., professor, biological chemistry, University of California, Irvine have developed a vector toolkit for systematic pathway engineering in S. cerevisiae.
Although scientists often study the introduction of individual genes using vectors and PCR-based integration, metabolic engineering typically requires the regulated expression of multiple genes to better optimize both pathway expression and function. To avoid instability, the integration of multiple expression cassettes may be required.
Drs. DaSilva and Sandmeyer and colleagues constructed a set of expression vectors for metabolic engineering applications. To promote differential expression of genes, six different promoters and six different reusable selection markers were included. The vectors were designed to allow the seamless transition from plasmid-based expression to PCR-based chromosomal gene integration.
Expression from the vectors and from multiple different integration sites has been characterized. According to Dr. DaSilva, the methodology can facilitate rapid and systematic combinatorial expression of pathway genes.
Deriving monoclonal antibodies (mAb) for therapeutic development can be like finding a needle in a haystack. Masaharu Isobe, Ph.D., professor of molecular and cellular biology, life sciences and bioengineering, University of Toyama, has developed a semi-automatic way for high-throughput generation of mAbs.
Quick mAb Production
“Although single-cell immunoglobulin variable gene cloning is the best way to generate recombinant monoclonal antibodies, these methods remain an obstacle to the rapid and high-throughput production of mAbs, particularly because of difficulties in stable amplification of the V genes from single cells and tedious cloning steps to obtain proper immunoglobulin gene-expression constructs.
“We developed a novel overlap extension PCR methodology in conjunction with a new instrument for 5´RACE-ready cDNA synthesis that produces a large number of recombinant mAbs very quickly.”
Dr. Isobe’s group developed a robotic magnetic bead-handling instrument. “Single cell-based cDNA synthesis is a required fine art due to the limited amount of source. To automatize cDNA synthesis from large numbers of single cells, we constructed a noncontact magnetic power transmission instrument that we call the MAGrahder.
“It has MAGrahder reactor trays and a desktop robot. It also has 12-channel, parallel magnetic rods on a robotic arm that transports and mixes nucleic acid-bound magnetic beads in the trays. This is followed by mRNA extraction, reverse transcription, and the homopolymer-trailing reaction that can handle up to 144 samples and only takes an hour to complete.”
However, according to Dr. Isobe, a second challenge was the need for a simple and efficient way to make expression constructs. Dr. Isobe’s method, called target selective joint polymerase chain reaction (TS-jPCR), amplifies gene fragments and assembles them into an immunoglobulin-expression construct without any purification steps.
“We join the 3´-random nucleotide-tailed V gene fragments produced by PCR with a specific immunoglobulin cassette containing all the necessary elements for antibody expression. The resulting immunoglobulin-expression constructs from amplified V genes are ready to transfect into cultured cells without any purification, and the media is used for the analysis of binding specificities of mAbs,” says Dr. Isobe.
“Our new system significantly reduces the cost and the time for generation of mAbs and allows us to obtain hundreds of mAbs within four to five days.”