DAIRY BIOTECHNOLOGY

Lesson 23. LARGE SCALE PRODUCTION AND DOWNSTREAM PROCESSING OF RECOMBINANT PROTEINS

Module 5. Application of biotechnology in dairying

Lesson 23

LARGE SCALE PRODUCTION AND DOWNSTREAM PROCESSING OF RECOMBINANT PROTEINS

Once a transformant or recombinant clone is selected based on detection of expressed protein by any of the above methods, the conditions for maximal expression of recombinant proteins are standardized. Further scale up is then carried out in fermenters (Fig. 23.1).

Fig. 23.1 Fermenter from applicon

Even the industrial micro-organisms are grown under controlled conditions with an aim of optimizing the growth of the organism or production of the target microbial product at industrial level. Fermentation is carried out in vessels called fermenters which have complex integrated system of automatic controls of pH, temperature, oxygen demand etc. Fermentation in liquid media is generally of three types depending upon the mode of operation viz. Batch Fermentation, Fed-batch Fermentation and Continuous Fermentation.

23.1 Batch Fermentation

It is the simplest mode of operation. The reactor is filled with the growth medium and fermentation is allowed to proceed. After inoculation, no medium is then added. After completion of fermentation, reactor is emptied and refilled again for new batch of fermentation.

23.2 Fed-Batch Fermentation

This is most widely used process in industries and is a modification of Batch type fermentation only. The reactor is filled with the growth medium and fermentation is then allowed to proceed. However, medium is periodically added after inoculation as the feed added at the start of fermentation gets exhausted. The product is obtained periodically and as well after completion of the fermentation process.

23.3 Continuous Fermentation

In continuous fermentation, fresh medium is supplied continuously. Products and cells are continuously removed for processing so that cells receive fresh medium continuously. It can be operated for longer periods. Growth rate of the cells can also be optimized by controlling the flow rate of the feed entering the reactor thus resulting in high productivity

23.4 Downstream Processing and Purification of Recombinant Proteins

Downstream processing refers to the recovery and purification of proteins/products either from

• Natural sources such as animal or plant tissue.
• Fermentation by wild micro-organisms or genetically modified

The products range from antibiotics, enzymes, hormones, therapeutic antibodies used in diagnostics, vaccines and several other recombinant proteins.

If the protein is intracellular, recovery involves the following steps.

i) Cell harvesting by centrifugation :

Fermentation process results in Biomass containing cells which must be separated for further processing. The separation of microbial cell normally involves either Filtration or Centrifugation.

23.4.1 Filtration

Filtration is generally used in case of very small sized microbial cells. It is useful for industries being cheapest option since slurry/microbial biomass can be fed directly to filtration unit attached with the output of the bioreactor.

23.4.2 Centrifugation

It is used in laboratory scale applications wherein cell biomass is separated by applying centrifugal force using a centrifuge (Fig. 23.2).

Fig. 23.2 Centrifuge from sanyo

i) Cell disruption generally involves ultrasonication, bead beater, microfluidizer and cell disruptor etc. for recovery of intracellular proteins

ii) Purification of the required product from cell extracts or the culture supernatant

iii) Confirmation of the target protein using SDS/PAGE, western blot or enzymatic assay

If the protein is extracellular, the recovery involves:

i) Concentration of the fermented medium either by dialysis, concentrator or ultra filtration.

ii) Purification of required product from cell extracts or the culture supernatant

iii) Confirmation of target protein using western blot or enzymatic assay

23.4.3 Ultrafiltration

Concentration involves passing of supernatant either from intracellular or extracellular extract through membranes of different cutoff values i.e. pore sizes (3, 5, 10, 30 or 100kD etc.). Molecules larger than the membrane pore size rating will be retained at the surface of the membrane. Hydrostatic pressure is applied that forces the liquid against a semipermeable membrane and this process is widely used in industry. For smaller volumes, centricons and ultrafiltration unit (Fig. 23.3) are used while for larger volumes tangential flow cartridge is preferred.

Fig. 23.3 Ultrafiltration unit

23.4.4 Chromatography

23.4.4.1 Gel filtration

Gel-filtration chromatography separates molecules on the basis of size (Fig. 23.4). It involves passing a protein solution through a column which is packed with beads of a hydrated insoluble material (matrix) such as dextran, agarose or polyacrylamide. Larger molecules cannot enter the beads, hence come out first in void volume. On the other hand, smaller molecules enter the beads and take more time for elution.

Fig. 23.4 Gel Filtration chromatography

23.4.4.2 Ion exchange chromatography

Ion-exchange chromatography separates molecules based on differences in net charge (Fig. 23.5, 23.6 and 23.7). Proteins with a net positive charge will be retained on negatively-charged columns such as carboxymethyl-cellulose (CMC) and those with a net negative charge will be retained on positively- charged columns such as diethylaminoethyl-cellulose (DEAE-cellulose).

Fig. 23.5 CMC and DEAE groups
chromatography

Fig. 23.6 Ion exchange

Fig. 23.7 Ion exchange chromatography

23.4.4.3 Hydrophobic interaction chromatography

Hydrophobic word is assigned to molecules that distracts water. Hydrophobic Interaction Chromatography (HIC) is a; liquid chromatography used to separate proteins on the basis of relative hydrophobicity wherein hydrophobic ligands bind to hydrophilic ligands on the separation matrix. Hydrophobic amino-acids can interact with HIC gel under high salt concentrations and then to bring about desorption, the salt concentration is lowered gradually and the proteins elute based on their hydrophobicity.

23.4.4.4 Reversed phase

Reversed Phase Chromatography (RPC) separates molecules according to differences in their hydrophobicity. RPC has become increasingly important for high-resolution separation and analysis of proteins, peptides and nucleic acids.

23.4.4.5 Affinity chromatography

Affinity chromatography is a purification technique that offers purities >95% in just one step. It makes use of a specific native or added property of the target molecule to isolate it from all other contaminants in the sample and is most popularly used in separation of recombinant protein in one step. Recombinant proteins can be purified using affinity columns since most of them are expressed as fusion proteins which helps in quick separation of proteins from several other proteins. Most common fusion partners include Glutathione – S- transferase (GST), Thioredoxin, Maltose binding protein and six histidine tags (His6) which use glutathione, phenylarsine oxide, amylose and nickel respectively (Fig. 23.8). One of the most common affinity chromatography technique is Immobilized metal ion adsorption chromatography (IMAC) also known as metal chelate affinity chromatography. IMAC is an excellent chromatography technique for optimization and purification of histidine tagged proteins since most of the vector systems used for expression of recombinant proteins have six his tag. The bound proteins can then be eluted by competitive elution with, for example, imidazole, or by lowering pH. Strong chelating agents, such as EDTA can also be used.

Fig. 23.8 Separation of protein using His tag affinity chromatography

23.4.5 Isoelectric focussing

Every protein has a unique isoelectric point, a pH at which the net charge on the molecule becomes Zero. In presence of ampholytes and a charged field, protein molecules migrate till the net charge on them becomes nil, thus resulting in efficient separation. It does not require denaturation of proteins thus maintains their biological activity. Rotofor (Bio-rad, Fig. 23.9) is commonly used for separation of proteins based on their isoelectric point.

Fig. 23.9 Rotofor from Bio-Rad

23.5 Confirmation of Identity of Recombinant Protein Using SDS-PAGE, Western Blot or any Other Enzyme Assay Available for a Particular Gene

After purification of the recombinant protein already described above from fermentation broth, SDS-PAGE is run to confirm the purity as well as molecular weight of the protein with the help of western blotting, ELISA or protein/enzyme assay etc.

23.6 Production of Specific Recombinant Enzymes/Proteins of Commercial interest for application in dairy/food industry

The detailed protocols for the production of two industrially important proteins having prospects for application in dairy industry are given below.

23.6.1 Buffalo chymosin

Chymosin is an aspartyl protease (Fig. 23.10) secreted in the fourth stomach (abomasum) of the suckling ruminants. Bovine (cattle) calf chymosin popularly known as calf rennet is the milk clotting enzyme present predominantly in the cheese rennet which has been traditionally used as the key ingredient for cheese making in the dairy industry and the typical cheese flavor is principally attributed to the activity of this enzyme. Chymosin specifically recognizes the k-casein sequence from amino acid position His98 to Lys111 and cleaves peptide bond between Phe105 - Met106 in casein.

Fig. 23.10 Primary structure of bovine chymosin

Traditionally, calf rennet is derived from the abomasal tissue of the suckling bovine calves after slaughter. However, the production of bovine chymosin is now greatly limited due to decreasing bovine calf population all over the world. This situation coupled with tremendous growth of cheese industry has considerably accelerated the demand for calf rennet in cheese industry. Hence, to meet this growing demand for cheese manufacture, concerted efforts are now being directed to find appropriate substitutes for bovine calf rennet. Although milk clotting enzymes from microbial sources have found wide applications in dairy industry, the quality of processed cheese using these enzymes has been inferior to that made from cattle calf rennet. Such products have limited consumer acceptability due to bitter flavor produced in the cheese. However, chymosin from abomasum of young buffalo calves could serve as a very attractive substitute for calf rennet as it has been reported to possess higher milk clotting activity compared to cow, goat and porcine chymosin. In addition, the chymosin from buffalo is expected to have an inherent compatibility with buffalo milk and is better in milk clotting activity than chymosin from heterologous sources. Therefore, the use of buffalo chymosin could be a preferred option for processing buffalo milk into common cheese varieties like cheddar and Mozzarella especially in countries like India with large population of buffaloes. India also harbors a major population with vegetarian food habit. As a result of growing public awareness, cheese produced using calf rennet is not acceptable by the vegetarian consumers due to religious sentiments. Hence, concerted efforts have been made in the last decade to explore biotechnological interventions to produce recombinant chymosin from sources other than cattle through heterologous expression of chymosin gene. The buffalo recombinant chymosin will not only provide a viable and cheap substitute for calf rennet but could also cater to the needs of vegetarian population by producing vegetarian cheese varieties specifically Mozzarella cheese extensively used in the preparation of Pizza. NDRI scientists at Molecular Biology Unit, Dairy Microbiology Division have expressed goat chymosin in E. coli and buffalo chymosin in Pichia pastoris - a methylotropic yeast.

23.6.1.1 Expression of goat chymosin in E. coli

Goat prochymosin cDNA was cloned and characterized by sequence analysis. The prochymosin cDNA spanned 1101 nucleotides and was predicted to code for 365 amino acids with a proregion of 42 amino acids. The cDNA fragment containing goat prochymosin was then subcloned in to pET43.1a (+) and expressed as a NusA fusion protein in E. coli which showed low level of milk clotting activity after activation at acid pH.

23.6.1.2. Expression of buffalo chymosin in E. coli

Buffalo chymosin was also cloned, sequenced and expressed in E. coli. The expressed protein has been shown on SDS-PAGE in Fig. 23.11. However, the expression level was not adequate and cost effective for its large scale production. Hence, further attempts were made to express buffalo chymosin in Pichia pastoris.

Fig. 23.11 SDS-PAGE showing expression of buffalo chymosin

23.6.1.3 Expression of buffalo chymosin in pichia pastoris

The strategy used to clone buffalo prochymosin insert from E. coli to Pichia pastoris vector pPICZαA has been outlined below in Fig. 23.12. After ensuring the in frame insertion of buffalo prochymosin insert, the construct was digested with SacI for linearization and electroporated into Pichia pastoris host X-33. Further to this, expression studies were carried out to study the level of expression in shake flask and various parameters were attempted to optimize the expression level. The expressed protein chymosin has been shown on SDS-PAGE (Fig. 23.13).

Fig. 23.12 Cloning strategy of buffalo chymosin in pPICZα

Fig. 23.13 SDS-PAGE showing recombinant buffalo chymosin

Buffalo chymosin has been expressed in the culture supernatant of P. pastoris in a 10 liter fermenter (Fig. 23.14) and production conditions and down steam processing have been optimized for the recovery of the recombinant product. The expression of the protein has been achieved to the level of 200-300 mg recombinant protein/L. Partially purified recombinant buffalo chymosin was successfully used as a milk clotting enzyme in the production of Mozzarella cheese (Fig. 23.15) from buffalo milk and the quality of the cheese made with recombinant enzyme has been comparable with that of cheese made with Meito rennet.

Fig. 23.14 Production of recombinant buffalo chymsoin in 10L fermenter

Fig. 23.15 Mozzarella cheese prepared using recombinant buffalo chymosin

23.6.1.4 Applications

• Production of all varieties of cheeses

• Production of casein hydrolysates

• Pizza market through production for Mozzarella cheese

23.6.2 Human lactoferrin

Lactoferrin – a naturally occurring unique glycoprotein found in milk of mammalian species has been receiving increasing attention and interest due to its multiple bioactive functions beneficial for human health. It is a unique multifunctional protein expressed in the milk of mammalian species such as the cow, buffalo, pig, equine, goat and mouse and particularly human. Its major functionality is related to its role in iron absorption and strong iron-binding properties. Lactoferrin is also used in the treatment of bacterial, viral and fungal infections, sepsis, cancer, tumors and immunosuppressory illnesses both in human and veterinary medicine. Human lactoerrin (hLf) with several bioactive functions in particular can find potential market in health / functional foods for elderly or immuno-compromised hosts for recovery from gastro-intestinal infections, and prophylactic products for traveler’s diarrhoea as well as can address the problem of antibiotic resistant strains especially methicillin resistant staphylococci. Lactoferrin fortified dairy foods will also have extended shelf life and hence can find potential applications in dairy, food and meat industry. Alternatively, the iron-loaded lactoferrin can be added to foods such as baby formula, cereal, and ice cream to enhance the nutritive value of the food. However, it is impractical to purify native lactoferrin particularly from human milk in order to make it a commercially viable product. Nevertheless, worldwide production of bovine lactoferrin (Lf) has increased tremendously during the last decade. Currently, for commercial production of Lf, bovine colostrum is used and Lf is isolated from cheese whey or from skim milk. Due to minute concentration of Lf in bovine milk, the recovery is too low that adds to the cost of production thereby making it commercially nonviable.Amongst the different MNCs, DMV is marketing bovine lactoferrin. However, bovine lactoferrin does not have the same biological effect since it has lower affinity for human lactoferrin receptors. Hence, NDRI scientists at Molecular Biology Unit expressed the same in two of the yeast systems namely Saccharomyces cerevisiae and Pichia pastoris. The cloning strategy was similar to that used for chymosin as described previously for chymosin. Human lactoferrin (hLf) cDNA was cloned and sequenced both from mammary gland tissue and neutrophils and finally expressed in Saccharomyces cerevisiae and in Pichia pastoris. Recombinant human lactoferrin is being currently produced by Agennix, a Houston based US Biopharma company and Ventria Bioscience, a California based company. The Agennix has expressed human lactoferrin in Aspergillus and Ventria exploits their ExpressTec for expression of Lf in rice.

Human Lactoferrin has been expressed intracellularly in S. cerevisiae at 5 mg/l level. The yeast biomass as such can be incorporated into food products which will serve as the source of lactoferrin as well as vitamins. The yeast host has GRAS status. Human lactoferrin has also been expressed in Pichia pastoris at a level of approximately 10-20 mg/l in a 10 L fermenter. Total protein was calculated to be 6.3 mg/l in supernatant and about 12 mg in cell pellet. The recombinant lactoferrin has been shown in Fig. 23.16.

Fig. 23.16 Recombinant human lactoferrin

23.6.2.1 Application of human lactoferrin

a) Pharmaceuticals

• Stimulation of immune system, regulation of iron metabolism, control of cell or tissue damage and as an antioxidant, prevention of osteoporosis, vaginal candidiasis and treatment of peptic ulcers.

• Treatment and prevention of opportunistic bacterial, viral, and fungal infections such as pneumonia, acquired immune deficiency syndrome (AIDS), candidiasis, diarrhoea, and neonatal sepsis.

• Anticarcinogenic - treatment of tumors such as brain tumors.

• Lactoferrin can aid in reducing heart attacks.

• Against Dental caries.

• As an Anti-inflammatory drug

b) Functional / Health / Nutraceutical Foods

• Lactoferrin can have potential market in health / functional foods for elderly or immuno-compromised hosts, recovery from gastro-intestinal infections, and prophylactic products for traveler’s diarrhoea as well as to address the problem of antibiotic resistant strains especially methicillin resistant staphylococci. Lactoferrin fortified dairy foods will also have extended shelf life.

• As nutritional supplements that include tablets, gelatin capsules, or liquids containing the lactoferrin together with adjuvants or diluents.

• Alternatively, the iron-loaded lactoferrin can be added to foods such as baby formula, cereal, and ice cream to enhance the nutritional value of the food.

• As a food preservative in food and meat industry.

c) Other Applications

• As an antiseptic either alone or in the form of a powder, solution, ointment, aerosol spray, or cream.

References

Protein purification. Philip L. R. Bonner. Taylor & Francis.
Protein purification: Principles, high resolution methods and applications. Jan-Christer Janson. John Wiley and Sons. 2011