Creative Enzymes provides custom services for expression and production of both natural and recombinant enzymes in various systems. As understanding of enzymology and molecular biology progresses, enzymes have been found more and more important in almost all biological pathways. At this point, there are over 200 protein-based pharmaceuticals licensed by FDA. Sales of therapeutic enzymes and industrial enzymes summed to over $6 billion by 2010. Rapid growth in the market demands and product development requires services of large-scale production. Meanwhile, new enzymes are being developed to meet all types of needs in pharmaceutical and catalysis. Advanced expression of enzymes with special properties and functions is desired by all researchers. Creative Enzymes established and developed expression systems for all types of enzymes, covering early stage of cDNA preparation, DNA cloning, enzyme expression, all the way to production scale-up. Our outstanding scientists and strategic partners professionally handle every detail of each step. We are the experts you can trust in the whole process of enzyme expression.

Several steps are necessary to obtain a natural or recombinant enzyme depending on the source of the gene sequence and the specific technical requirement: The initial cDNA synthesis, amplification, and isolation; followed by insertion into a vector; and lastly induction and expression of the target enzyme (Figure 1). The first two steps are also usually considered as cDNA cloning. A desired enzyme could be a natural enzyme existing in high abundance in specific cells at a certain time, which is generally related to high abundance of the corresponding mRNA. Therefore, the mRNA can be identified and isolated to be used as the template for reverse transcription to generate cDNA, which is complementary to the mRNA sequence and does not contain introns or operons. Alternatively, if the coding sequence of the enzyme is known, fully synthesize cDNA sometimes is a fast and more economic approach. After obtaining the target cDNA, amplification of the cDNA copies is carried out. Traditionally, a cDNA copy is inserted into a vector and host cells are used to multiply the vector DNA, also known as recombinant DNA, along with the DNA of the host cells. The subsequent isolation and purification will give the target cDNA in a large quantity. However, multiple steps of cutting and ligation, as well as purification of DNA are involved in this process, resulting in high cost and low efficiency. Thus, the PCR (polymerase chain reaction) technology has revolutionized cDNA cloning since it was invented. The technology makes DNA cloning much faster and easier for both particular DNA clones and construction of a cDNA library. With recent development of the technology, such as qPCR (quantitative PCR) and RT-PCR (reverse transcription PCR), it has become the most commonly used and often indispensable tool in cDNA cloning.

Figure 1. A scheme of simplified cDNA cloning and expression.

To produce a recombinant enzyme from the cDNA copy, an expression vector needs to be chosen, with the consideration of choosing proper expression host suitable to the vector. Although no specific priority is given in the selection of expression vectors, several key factors should be considered. First, the selected vector must have high plasmid stability throughout fermentation. Second, the origin of replication should ensure the production of a high copy number of plasmid, which is also important to large scale production. Next, the vector should contain multiple cloning sites or a cloning region where the target DNA can be easily ligated into. In addition, the transcription promoters and fusion tags should be also considered when choosing the vector. Alternatively, Creative Enzymes also offers auto-induction media, which requires little human intervention during fermentation. The technology is based on E. coli metabolic control and automatically performs a shift from growth to recombinant protein expression at the proper time point. The advantages are ideal for the large-scale production of recombinant enzymes.

When it comes to expression hosts, a wide variety of protein expression systems are available. Enzymes can be expressed in cell cultures of bacteria, yeasts, molds, mammals, plants or insects, or via transgenic plants and animals. When choosing the expression system, protein quality, functionality, production speed and yield are the most important factors to consider. Non-glycosylated proteins are usually made in E. coli or yeasts, the most common expression systems. N-glycosylated proteins are usually produced in mammalian cells which mimic human glycosylation, which is especially important to therapeutic enzymes. For example, about half of the therapeutic protein market is provided with Chinese hamster ovary (CHO) cells. However, the process is expensive and the glycoproteins could still be different from the human type, and thus sometimes needs further modification. Yeasts, molds and insect cells are also genetically engineered to produce a human type of glycosylation. Generally, bacteria and yeasts are the most developed systems, which means lost-cost expression and production; while higher organisms are more expensive to operate but also provide the capability to product more complex enzymes (Figure 2). At Creative Enzymes, we always suggest to first try an E. coli strain because of their ability to grow rapidly and at a high density, low cost and high productivity. However, if the researcher experiences one of the drawbacks of the system, such as the formation of inclusion bodies, incorrect protein folding, and degradation of the heterologous protein, especially when the heterologous enzyme is originally isolated from prokaryotes, other expression systems, such as yeast, insect and mammalian systems should be considered.

Figure 2. Advantages of typical hosts for enzyme expression.

With decades of experiences in cloning and expression, Creative Enzymes offers several unique and advanced solutions for many types of enzymes. As one could expect from the previously mentioned key points of expression system selection, choosing the most suitable system for an enzyme greatly relies on the experience of the researcher, as well as thorough consideration of the enzyme properties. Therefore, we encourage the customer to share as many details as possible when submitting the service request. With many years of excellent service in the global market, we believe you will be fully satisfied with the professional and productive services. Please do not hesitate to contact us for a quotation of our expression and expression-related services:

• Expression evaluation and optimization
• Bacteria
• Escherichia coli
• Bacillus
• Other bacteria
• Yeasts
• Saccharomyces cerevisiae
• Methylotrophs, such as P. pastoris and H. polymorpha (P. angusta)
• Other yeasts
• Fungi
• Baculovirus/Insect cells
• Mammalian cells
• Transgenic animals
• Transgenic plants
• Cell free expression (in vitro expression)
• Difficult-to-express enzymes
• Toxic enzymes
• Unstable enzymes
• Peripheral enzymes (membrane protein)
• Cell line/host development
• Production scale-up