Genetically Engineered Plants as Protein Factories: A Close View

Read this article to learn about metabolic engineering of proteins in plants.

The genetically engineered plants can manufacture a wide range of proteins such as: (1) Industrial Enzymes (2) Lysosomal Enzymes (3) Antibodies (4) Vaccines and (5) Therapeutic Proteins.

Molecular farming or metabolic engineering of proteins in plants is a novel approach that makes plants to serve as living factors (bioreactors) for the production of proteins. Use of animals, animal or microbial cell cultures for the bio-production of proteins has been described .Production of human or animal proteins by plants is a more recent and novel approach.

The transgenic plants have the following advantages as bioreactors:

i. Low cost of production.

ii. Eukaryotic protein processing.

iii. Unlimited supply.

iv. No spread of animal borne diseases.

v. Reduced time to market.

vi. Safe and environmental friendly.

vii. No need for skilled workers.

Genetically engineered plants can manufacture a wide range of proteins. These include:

i. Enzymes for industrial and agricultural purposes.

ii. Lysosomal enzymes.

iii. Antibodies (plantibodies).

iv. Subunit vaccines.

v. Other biopharmaceuticals or medically related proteins.

The general approaches for the production of desired proteins in plants are first described, followed by the different products of commercial importance.

Approaches for Protein Production in Plants:

The general methodology for the production of proteins in plants involves the following aspects:

1. Selection of crop species.

2. Choice of tissue.

3. Expression strategies.

4. Post-translations processing.

5. Recovery strategies.

Selection of crop species:

The most important consideration for choosing a plant as a bioreactor is that it should be readily manipulated to produce a stable transgenic line, with stable expression.

Tobacco-the most preferred plant as a transgenic bioreactor:

Tobacco is the most widely used plant for the bio-production of proteins by researchers as well as biotech companies, for the following reasons.

1. It can be easily engineered and transformed.

2. Tobacco is an excellent biomass producer (about 40 tons of fresh leaf/acre land against 4 tons by rice).

3. Seed production is very high (about one million seeds/plant).

4. Tobacco can be harvested several times in a year.

The major limitations of tobacco plant are — it is a regional crop and relatively labour intensive, and it is not suitable to be fed to humans or animals.

Other crop species as bioreactors:

For the development of edible products (e.g. vaccines) plants such as potato, tomato, banana, maize and lettuce are used. Some workers prefer to produce proteins in the seeds of plants such canola, soybean, corn, rice and barley. This is mainly because seeds can accumulate and store good quantities of proteins.

Choice of tissue:

The preferred tissues for protein production by plants include leaves, storage organs (e.g. tubers) and seeds.

This choice of tissue mainly depends on the following factors:

i. Compatibility with the desired protein.

ii. Correct processing.

iii. Stable accumulation.

iv. Efficiency of recovery.

Many proteins have been produced in leaves (particularly using tobacco plant) e.g. lysosomal enzymes, mammalian antibodies, human serum albumin. In recent years, seeds have become an attractive high protein production (e.g. hirudin) vehicles due to good storage capacity and stability. It is no surprise that some of the seed proteins retain their biological activity for several years. One limitation of seeds is that it is sometimes difficult to recover the protein.

Expression strategies:

There are two major approaches for the transgene expression in plants to produce proteins- stable integration approach and use of plant viruses as transient vectors.

1. Stable integration approach:

This strategy is most suitable for the bulk production of proteins particularly in leaves. In this approach, the transgene is regulated by a strong constitutive promoter such as 35S promoter. Another alternative is to use tissue-specific promoter.

By restricting the transgene expression to a particular tissue (e.g. seed), the yield of the protein can be substantially increased. In addition, the protein is stable for a long time from weeks to years. A diagrammatic representation of stable gene expression approach for protein production is depicted in Fig 51.4.

2. Transient expression by using plant viruses:

Bio-production strategies involving viruses are useful for the protein production at the desired period (discrete period). By genetic manipulations, it is possible to insert the desired gene (for protein) into the coat protein gene of the virus genome. The so developed infectious RNA viruses are introduced into the plants for the production of proteins.

Transient expression system by viruses has been successfully used in tobacco plants by employing tobacco mosaic virus (TMV). Tobacco plants at an appropriate age were inoculated with genetically modified TMV. Recombinant protein could be extracted within 2-3 weeks of harvesting.

Post-translational processing:

The ultimate purpose of the use of plants as bioreactors is to produce proteins with native conformation, good biological activity and biocompatibility. The specific folding of the protein with disulfide bonds and suitably modified amino acids is important for this purpose.

The changes that occur after (or sometimes even during) the protein is synthesized are collectively referred to as post-translational modifications e.g. phosphorylation, hydroxylation, carboxylation, glycosylation, proteolytic processing etc. Variations in post-translational changes are often limiting the successful production and use of recombinant proteins either in plants or even in other systems (transgenic animals or cell cultures).

The major limitations of plants with regard to post-translational modifications of proteins are listed:

i. Glycosylation (incorporation of carbohydrate moiety) is different in plants.

ii. Plant glycosylation results in the production of complex glycans containing fucose or xylose, which do not occur in humans.

iii. Plants cannot perform specialized carboxylation necessary for certain clotting factors (II, VII, IX, and X) and enzymes (protein C).

Due to above limitations, many a times proteins of transgenic plants are inefficient or less efficient in their function, besides exhibiting immunogenicity.

In recent years, cloning of genes responsible for post-translational modifications of proteins (e.g. glycation, carboxylation) have met with success to produce more biologically active proteins.

Recovery strategies (purification strategies):

A rational approach for a cost-effective purification strategy is desirable for the recovery of the proteins (downstream processing) produced in transgenic plants. The degree of purity is dependent on the purpose for which the protein is produced. For instance, industrial enzymes in general, do not require high degree of purity.

Sometimes, purification may not be necessary at all. A good example is the production of the enzyme phytase in seeds. Phytase acts on phytate and increases the availability of phosphate to animals. For this purpose, milled transgenic seeds containing phytase can be directly added to the feed. This totally avoids the purification/downstream processing costs.

For a great majority of proteins, particularly pharmaceutical proteins, purification strategy is absolutely required. It is therefore necessary to develop efficient methods for downstream processing. Some of the recovery strategies are briefly described.

Affinity tag-based purification:

The use of affinity tags for the purification of recombinant proteins has been described elsewhere and a similar strategy works in plant systems as well. The technique involves the fusion of desired protein with another protein or peptide (tag), which can bind to ligand, and thus the protein can be isolated. Following purification of the desired protein, the affinity tag can be removed. Some workers have been successful in purifying the enzyme glucocerebrosidase by this approach.

Purification through compartmentalization:

It is possible to direct the accumulation of proteins in a specific sub-cellular organelle. This is achieved by using signal peptides or fusion peptides. Once the protein is localized, the technique of sub­cellular fractionation can be used for preliminary isolation of protein followed by its purification. The human neuropeptide leueukephalin has been compartmentalized in the endoplasmic reticulum and vacuoles by this strategy. Then by appropriate purification techniques, it is isolated.

Protein purification by production in oil bodies:

Oil bodies are natural sub-cellular organelles that store triacylglycerol’s (TG). Oil bodies are composed of triacylglycerol’s enveloped by a phospholipid monolayer. A group of proteins, referred to as oleosins are associated with the surface of the oil bodies.

Oil bodies are found in the oil/seeds that represent the storage form of energy (as TG). Oil seeds contain oleosin at a concentration in the range of 2-10% of the total seed proteins. Oil bodies can be used for the production of foreign proteins in plants. The development of transgenic plants and the purification of the desired protein by employing oil bodies are shown in Fig. 51.5.

A transgene with oleosin and desired protein can be constructed. By introducing this gene, transgenic plants are developed. Oleosins tolerate foreign proteins; hence it is possible to produce oleosin-fusion proteins that accumulate in the oil bodies.

The plant materials (mainly seeds oil bodies) are crushed and centrifuged to separate into three fractions:

i. A pellet of insoluble plant material at the bottom of the tube.

ii. An aqueous phase containing soluble components of the seeds at the middle.

iii. A floating layer at the top with oil bodies and their associated oleosin fusion proteins.

The top layer of oil bodies is isolated, re-suspended in a buffer and re-centrifuged. The concentrated oil bodies are treated with the proteolytic enzyme protease that cleaves the desired protein from oleosin. On further centrifugation, the pharmaceutically important protein (aqueous phase) can be separated from the oil bodies.

Oleosin Partition Technology:

The purification of foreign proteins through their production in oil bodies is referred to as oleosin partition technology (Fig 51.5). The oil bodies along with the oleosin- fusion proteins are quite stable within the seed and during the course of processing. The pharmaceutical protein of interest remains stable retaining its biological activity within the seeds for years.

Further, the purification oleosin-fusion proteins is relatively easier. Hence oleosin partition technology is preferred for the purification of many pharmaceutically important proteins. Two functional proteins namely hirudin and β-glucuronidase have been recovered by oleosin partition technology.

Production of hirudin in Brassica napus:

Hirudin is a natural anticoagulant protein produced in the salivary glands of leeches (Hirudo medicinalis). It is an effective therapeutic agent as it specifically inhibits thrombin and blocks blood coagulation. Till some time ago, hirudin was produced by employing bacteria and yeasts. The annual requirement of hirudin is very high, and it is a good candidate for producing in transgenic plants.

A synthetic gene encoding hirudin was fused with Arabidopsis oleosin gene. An endopeptidase factor Xa gene was inserted between hirudin and oleosin genes. (The endopeptidase gene serves as a recognition site for the protease action). The transgene construct was introduced into Brassica napus. The so developed transgenic plants produce oil bodies containing oleosin-hirudin fusion protein. Hirudin can be purified by the procedure described already and depicted in Fig 51.5.

Oleosin partition technology holds a great promise for the successful commercial production of several pharmaceutically important proteins.

Production of Industrial Enzymes in Plants:

There is an ever-growing demand for industrial enzymes. Existing sources may not be able to meet the requirements. Large-scale production of plant- derived transgenic enzymes for commercial and industrial purposes are preferred.

Besides several advantages of plants as bioreactors, there is no fear of spread of animal pathogens (like BSE) as is the case with animal production. A selected list of industrial enzymes produced in transgenic plants and their important applications is given in Table 51.2. Some of the important enzymes are described.

Avidin and β-glucuronidase:

The first proteins/enzymes that were produced in transgenic plants (maize) are avidin and β-glucuronidase. They are used in diagnostic kits.

Trypsin:

Trypsin is an important proteolytic enzyme and its production by conventional recombinant approaches is rather difficult. Trypsin is now produced in plants. It has a wide range of applications-for the production of insulin, vaccines, wound care etc.

Phytase:

Phytase is a hydrolytic enzyme that catalyses the hydrolysis of phytate (inositol hexaphosphate) to inositol and inorganic phosphate. Phytate is present in high quantities in many plant seeds used as a feed to pigs and poultry (monogastric animals). These animals do not possess the enzyme phytase; hence they cannot derive the nutrient phosphate from phytate. The undigested phytate gets excreted and accumulates in the soil and water, leading to eutrophication.

Transgenic plants capable of synthesizing phytase in their seeds have been developed. These seeds (no need to isolate the enzyme) can be mixed with the feed of animals, and fed. The phytase enzyme has successfully solved nutritional (phosphate) and environmental (eutrophication) problems.

Cellulose and xylanase:

The two enzymes namely cellulose and xylanase are used in several industries- bioethanol, textile, paper and pulp. In all these processes, they are basically involved in the degradation of plant materials (predominantly cellulose).

By a novel approach, it is possible to produce cellulose and xylanase in the plants for which the target itself is the digestion of plant cell. The risk of autodigestion of the plant cells (by these enzymes) is overcome by genetic manipulation to produce thermostable enzymes.

Thus, cellulose and xylanase, produced by transgenic plants, are inactive at the temperatures at which plants normally grow. The activity of these enzymes is restored on heating the plant extracts. And these extracts are very successfully used in many industries.

While the recovery of enzymes from transgenic plants is often required, there are some instances where the crude extracts can be directly used. The industrial applications of cellulose, xylanase and phytase (described above) are some good examples.

Production of Lysosomal Enzymes in Plants:

Lysosomes are the cellular organelles responsible for the degradation of macromolecules. The degradative enzymes present in lysosomes include proteases, nucleases, lipases, glycosidases, phosphatases, phospolipases and sulfatases. Deficiency of a specific lysosomal enzyme results in the accumulation of un-degraded substrate causing clinical manifestations.

Some examples of lysosomal disorders and the corresponding enzyme deficiencies are listed below:

i. Tay-Sachs disease — a-hexosaminidase.

ii. Gaucher’s disease — glucocerebrosidase.

iii. Hurler syndrome—iduronidase.

iv. Mucopolysaccharidoses (a group of diseases) — Several enzyme of glycosaminoglycan degradation.

A couple of lysosomal enzymes produced in transgenic tobacco plants are described below.

Glucocerebrosidase :

Glucocerebrosidase is a lysosomal enzyme that degrades glucocerebroside (predominantly produced when erythrocytes are destroyed due to aging or damage) to glucose and ceramide. The deficiency of this enzyme causes Gaucher’s disease, characterized by swelling of spleen, liver, and bone damage causing extreme pain.

Gaucher’s disease can be effectively treated by administering the enzyme glucocerebrosidase. At present, this enzyme is obtained from human placenta or from mammalian cell cultures, both being very costly. It is estimated that a single patient requires 10-12 tons of human placenta annually costing $100,000 to 400,000!

Glucocerebroside was the first lysosomal enzyme produced in plants (Nicotiana tobacum). The production process of this enzyme has been patented, and FDA approved it for use in humans. It is hoped that through the plant-based production of glucocerebrosidase (marketed as Cerezyme), the patients of Gaucher’s disease can be treated in a cost-effective manner.

α-L-lduronidase:

The deficiency of the enzyme iduronidase causes Hurler’s syndrome, the most common disorder of defective mucopolysaccharide degradation. Tobacco plants have been genetically engineered to produce α-L-iduronidase.

Limitations for the production of lysosomal enzymes in plants:

Both the enzymes referred above are glycoproteins (glucocerebrosidase and iduronidase). As described elsewhere appropriate glycation of animal proteins is not feasible in plants. This poses a major problem for the production of lysosomal enzymes in plants. Through a series of genetic manipulations, biotechnologists were successful in achieving the desired glycation for the production glucocerebrosidase and iduronidase.

Production of Antibodies (Plantibodies) in Plants:

Antibodies or immunoglobulin’s are the defense proteins produced in mammals. The use of plants for commercial production of antibodies, referred to as plant bodies, is a novel approach in biotechnology. The first successful production of a functional antibody, namely a mouse immunoglobulin IgGI in plants, was reported in 1989.

This was achieved by developing two transgenic tobacco plants-one synthesizing heavy y-chain and the other light K-chain, and crossing them to generate progeny that can produce an assembled functional antibody.

Production of secretory IgA (slgA):

Secretory IgA is an immunoglobulin that protects against dental caries produced by Streptococcus mutans. For the production of slgA, transgenic plants were developed to synthesize different subunits and then crossed to produce the functional antibody (Fig 51.6).

Four separate transgenic plants synthesizing four distinct pieces of antibody (H-, L-, J-chains and secretory component) were developed. The plants expressing H- and L-chains were crossed (cross 1) giving plants that produce IgA.

When these plants were crossed (cross 2) with plants expressing J-chain, the progeny produced dimeric IgA. The dimeric IgA synthesizing plants were then crossed (cross 3) with the plants expressing secretory component, the functional slgA could be produced.

Secretory antibodies have many advantages. They are resistant to proteolytic degradation; hence their yield in plants is substantially higher slgA are the predominant antibodies that protect against mucosal infections of microorganisms. They bind to antigens more effectively and offer good protection.

The secretory antibodies produced in transgenic plants have been tested on humans. When slgA was topically applied to teeth, it could prevent the colonization by Streptococcus mutants up to 4 months. This is comparable to the protection offered by immunoglobulin’s produced through hybridoma technology. This was possible despite some structural differences between plant bodies and monoclonal antibodies. It appears that there is no difference in the binding properties between the two types of antibodies.

Production of other antibodies:

Through genetic manipulations, it has now become possible to produce a wide range of antibodies in transgenic plants-whole antibodies, antigen-binding fragments, and single-chain variable fragment antibodies. Some examples of plantibodies and their applications are given in Table 51.3.

It is striking to note that despite certain differences in glycosylation of antibodies produced in plants and animals, the plantibodies are by and large comparable in their function with animal antibodies.

Production of Vaccines in Plants:

Vaccination is an effective approach for proper healthcare of millions of people in the world. Sometimes, the cost of the vaccines is coming in the way, and consequently, thousands of children become susceptible to preventable diseases.

Vaccines are designed to elicit immune response without causing the disease. In the conventional approach, vaccines composed of killed or attenuated disease-causing organisms are administered. This method is sometimes associated with side effects. With the advances in molecular biology, recombinant subunit vaccines that are effective in preventing the disease, without causing side effects have been identified.

There are many production systems for the synthesis of recombinant vaccines. These include mammalian cell cultures, yeast, bacteria and insect cell cultures, and transgenic animals. Transgenic plants provide an alternate system for the production of recombinant vaccines. The major advantage of vaccine production in plants is the direct use of edible plants tissues for oral administration. By use of edible vaccines or veggie vaccines (vegetable vaccines), the problems associated with purification- of vaccines can be totally avoided.

The stable or transient expression system can be used to produce vaccines in plants. Some examples of vaccines produced in plants are given in Table 51.4.

Edible vaccine:

Plant-based production systems are designed to provide locally available edible vaccines, at low- cost, for easy administration to millions of people. The need for use of edible vaccines arose from the fact that a large number of people in the developing world are the victims of enteric diseases. Edible vaccines provide mucosal immunity against infectious agents.

Choice of plants for edible vaccines:

Most of work on the production of vaccines in plants was initially carried out in tobacco plant that is not edible. These vaccines are now being produced in edible plants such as banana, tomato, and to some extent potato. For use in animals, the common fodder crops or food crops can be considered.

Some workers consider banana as an ideal system for the production of edible vaccines. This is mainly because it is grown in most parts of the world, and eaten raw. Another advantage is that children (to whom vaccines are most needed) like eating banana.

Delivery of vaccine to the gut:

Vaccines, being proteins, are likely to be degraded in the stomach. This is true to some extent. However, it is now known that the orally administered plant materials (edible vaccine) can induce immune response. The particulate materials of plants are more effective in this regard.

Food-based tablets to replace edible vaccines:

It is true that the edible vaccines may work well in the laboratory or under controlled conditions. There is a difficulty of dose adjustment when edible vaccines are consumed as a part of foodstuff. Some workers prefer to discontinue the direct use of plant materials, and prefer to opt for food-based tablets containing a known dose of vaccine. This approach is being applied to vaccines produced in tomatoes.

Limitations of edible vaccines:

Direct consumption of transgenic fruit or vegetable or food-based tablets may create some problems.

i. The risk of loss of vaccines by the action of enzymes in stomach and intestine.

ii. The possibility of allergic reactions as they enter circulation.

The future of edible vaccines:

Despite several limitations, biotechnologists see a great hope for the new wave of transgenic veggie vaccines. But it may take several years before the banana or tomato replaces the needle as a vehicle for vaccine delivery.

Production of Therapeutic Proteins in Plants:

Many of the products of industrial enzymes, lysosomal enzymes, antibodies and vaccines in transgenic plants actually represent therapeutic proteins or biopharmaceuticals e.g. lysosomal antibodies, vaccines.

In Table 51.5, a selected list of therapeutic proteins and the plants producing them, along with the applications is given.

The major limitations of protein production by transgenic plants are the low product yield and the difficulties associated with recovery. With improvements in genetic engineering and recovery processes, it is hoped that the production of therapeutic proteins will substantially increase in the coming years.

Chloroplasts in the production of therapeutic proteins:

Chloroplasts are capable of folding the foreign proteins, besides bringing out most of the post-translational modification. As the chloroplasts are inherited maternally, they will not spread via pollen to non-transgenic systems. This type of biotechnological approach will help to get clearance from the regulatory bodies of biotechnology.

Some of the therapeutic proteins produced in chloroplasts are listed:

i. Interferon’s

ii. Serum albumin

iii. Hemoglobin

iv. Pro-insulin

v. Monoclonal antibodies

vi. Protective antigen of Bacillus anthracis.