By Katharina Schoebi, Checkbiotech

The market for pharmaceutical proteins, such as insulin, is estimated to be worth about $10 billion yearly. In a few years, when patents on the oldest protein drugs expire, the industry will need to search for new and cheaper production techniques to counter increased competition. Biolex, a biopharmaceutical company, has now developed some methods for a faster and more cost-efficient protein production.

In medicine, pharmacology, biology and industry, there is a need for commercial production of valuable proteins. Proteins, such as recombinant or synthetic insulin, or industrial enzymes used to make stone-washed denim for jeans, can be made by living systems, such as microorganisms or cultures of mammalian cells.

However, there are some proteins, which cannot be produced in mammalian systems, because the proteins interfere with cell viability, cell proliferation, cellular differentiation, or protein assembly. Modern technologies make it possible to produce these mammalian proteins in plants. In plants, such proteins can be produced even in much larger quantities than in mammalian cells.

Since not all plant species are suitable for the production of proteins, researchers have to select the right species for commercial production of valuable proteins. Dr. Anne-Marie Stomp, from North Carolina State University, developed the first procedure to genetically engineer duckweed (Lemnaceae) for the production of therapeutic proteins. Dr. Stomp's strategy is to turn the weed's oddities to her advantage.

Duckweeds are small, free-floating, fresh-water plants, which are found all over the world. They are the most morphologically reduced plants known, but nevertheless, most duckweed species have all the tissues and organs of much larger plants, such as roots, stems, flowers, seeds and fronds. The inherent characteristics of the duckweeds facilitate low-cost, highly efficient bioprocessing for the recovery of commercial levels of recombinant proteins.

Duckweed has some advantages over the existing microbial or cell culture systems – and over other plant species. With its special growth habit, it is ideal for microbial culturing methods. By intensive culturing, duckweed reaches the highest rates of biomass accumulation per unit time – it can double in size every 24 to 48 hours by cloning itself. Each little green disk is an entire plant unit and an identical copy of its parent. 15 to 45 % of dry weight of the tiny weed consists of proteins. So, scale-up of the biomass necessary for commercial production of proteins is much faster and more cost efficient in duckweeds than in other cells.

As mentioned above, some proteins cannot be expressed in mammalian systems, because they interfere with some cell characteristics. Since there exists a large evolutionary distance between duckweed and mammals, it is unlikely that these proteins will interfere with regulatory processes in duckweed.

One significant benefit of using transgenic plants, is that plants do not transmit any known human viruses as mammalian cells do. Thus, they cannot be contaminated by infectious agents. In the end, this will help reduce the production costs for companies such as Biolex.

Unlike soybeans and tobacco, two often genetically modified plant models, duckweed grows in a variety of environments. This flexibility allows it to be grown in the ultra-clean, controlled conditions required for pharmaceutical proteins, as well the low-cost, large-scale production needed for industrial enzymes.

Although the efficiency of duckweed protein production is already far higher than the one of existing microbial or cell culture systems, Dr. Anne-Marie Stomp, Lynn Dickey and John Gasdaska from Biolex have invented, and patented, methods and compositions that will help to increase protein yields produced in duckweeds even more.

The invention also describes methods by which genetically engineered duckweed plantlets are directed to secrete the biologically active polypeptides. The induced secretion of the proteins facilitates the recovery of the proteins from fluid surrounding the plants.

"Biolex is only pursuing recombinant protein products for use as therapeutics," said Dr. Stomp. They have no products in on the market place, yet.

For genetic engineering, the DNA-sequence of interest is introduced in the plantlet. This can be done by bacteria called Agrobacterium tumefaciens, which naturally introduce foreign genes into the plant genome.

Another way to introduce a gene of interest in a plant cell is by ballistic bombardment, where the DNA-sequences of interest are placed on little gold-particles, and then shot into the plant cells.

Since duckweeds naturally clone themselves, it is possible that each duckweed clone could produce at least one of the subunits of the whole protein. When the different clones are cultured together, the secreted subunits could be assembled to form a functioning protein.

Yet, if needed, it is possible to produce only some special subunits of a protein. For instance a special subunit could be produced for industrial or chemical processes or for diagnostic, therapeutic, or vaccination purposes.

"With protein production by duckweed, people in the developing world will have some advantages", said Dr. Stomp. Edible vaccines could be made in duckweed, which could easily be grown in simple pools or ponds, without any high technology facilities.