Define and explain how recombinant proteins are made using recombinant DNA technology

Recombinant proteins are a new combination of genes that forms DNA. Recombinant proteins

are made from cloned DNA sequences which usually encode an enzyme or protein with known

function (Cusabio, 2018).

Recombinant DNA (rDNA) is a DNA strand that is formed by the combination of two or more

DNA sequences (Cusabio, 2018).

Using recombinant DNA technology, scientists are able to create new DNA sequences that

would not naturally exist under normal circumstances and environmental conditions. The

resulting recombinant DNA is put into a host cell, where it is expressed into a new protein,

which is the so-called recombinant protein. The recombinant DNA technology plays a critical

role in the production of recombinant proteins for pharmaceutical, medical, agricultural, and

other uses.

So to be able to create recombinant proteins, we use genetic engineering, also called gene

splicing or recombinant DNA technology. By putting human, animal or plant genes into the

genetic material of bacteria, mammalian or yeast cells, these microorganisms can be used as

factories or producers to make proteins for medical, academic and research uses (Cusabio,

2018).

Some researchers have summarized the several main steps in the production of recombinant

proteins, as follow:
 1. Obtaining the cDNA and creating the expression clone - the process of isolating a certain

DNA clone that contains the specific gene of interest

2. Cloning – the replication of recombinant protein to provide a subject for analysis,

sequencing, and others

3. Expressing the protein in a suitable system – laboratory approaches necessary for

producing proteins

4. Small-scale test expression – used to increase the protein’s chances to have a soluble

and active state as well as in high yield

5. Protein purification – to preserve the largest quantity of functional protein with few

contaminants as possible

6. Protein characterization – proteins’ differences in size, molecular structure, and

physiochemical properties entails for protein analysis by separation or identification.

Electrophoresis separation differentiate proteins by size or mass while in isoelectric

focusing, proteins are separated by charge

Apply recombinant DNA technology methods to describe how genetically engineered

chymosin is produced

First and foremost, what even is a recombinant DNA? rDNA is made through the combination

from two or more organisms. There are two methods in recombinant DNA: the Maxam-Gilbert

method, discovered by and named for American molecular biologists Allan M. Maxam and

Walter Gilbert, and the Sanger method, discovered by English biochemist Frederick Sanger. But
the most commonly used between the two is the Sanger method wherein there are three main

steps: (1) The DNA sequence of interest is used as template for Chain Termination Polymerase

Chain Reaction (PCR) to result oligonucleotide. (2) The terminated chains of oligonucleotide will

be separated by size through the process of gel electrophoresis and (3) the gel will be analyzed

to determine the resulting DNA sequence. (Griffiths, 2020)

But what is the relevance of this to how genetically engineered chymosin is produced? Firstly,

chymosin is found in a calf’s stomach and is responsible in coagulating milk and to produce

cheese. So, chymosin is genetically-engineered through recombination. Basically, the chymosin

gene from cows is obtained by gathering tissue from the cow and then isolating the DNA from

its cells which is then amplified through PCR. Afterwards, the DNA is introduced into a plasmid.

Then, the plasmid is inserted to a microorganism which will trigger the production of chymosin

by protein synthesis. The process needed to combine the chymosin gene and the microbe’s

which makes this a recombination process.

Evaluate the scientific, ethical, and social implications of using recombinant DNA technology

to produce proteins used in food preparation

In implementing recombinant DNA technology, it requires a scientific, ethical, and social

approach in order for it to completely work. The scientific implication plays a vital role in

development of science that makes human life much easier. In recent years, it has contained

advanced strategies for biomedical applications such as cancer treatment, genetic diseases,

diabetes, and several plant disorders especially in viral and fungal resistance. While in ethical
implications, it contains a review of autonomy, confidentiality, privacy, and equity. Among the

various definitions of privacy, one broad definition that captures its central element is that

privacy is a state or condition of limited access to a person. Every person has the right to his or

her own privacy, because once a person undergoes genetic tests, it includes the right of that

person to be informed and has an independent decision whether others may know more about

the details of their genome (e.g, insurers, employers, spouses, and family members). Also, in

social implication, it has proven to be essential to the production of vaccines and protein

therapies such as human insulin, interferon, and human growth hormone. However, it could

also create unknown side effects or outcomes. Certain changes vary from plants and animals

may cause unpredicted allergic reactions in some people, wherein it doesn’t occur in its original

form. In conclusion, knowing these factors or implications may help you gain more knowledge

of what you will be doing in order for you to prevent unnecessary actions or results to come in

the future.

What is Industrial Fermentation?

Industrial fermentation is the use of living organisms (especially microorganisms), usually on a

large scale in order to produce commercial products or carry out important transformations

such as chemical transformations. The goal of industrial fermentation is to enhance biochemical

or physiological processes that microbes (a microorganism, especially bacterium that is causing

disease or fermentation) are capable of performing while producing the highest form and

number of a certain product. The developmental stage of the fermentation process requires a
deeper thinking from disciplines such as microbiology, biochemistry, genetics, chemical and

bioprocess engineering, chemistry, mathematics, and even in the field of computer science. The

major microorganisms used in industrial fermentation are fungi (like yeast) and bacteria. In

addition to this, fermentation is performed in large bioreactors or fermenters often of several

thousand liters in capacity. Lastly, industrial fermentation is a part of many manufacturing

corporations such as microbiology, food, pharmaceutical, biotechnology, and chemical (M.,

Sergei, 2012).

What is genetic engineering and how it is used in industrial fermentation?

Sometimes called genetic modification or generic manipulation. It is the process of altering the

DNA in an organism’s genome using biotechnology. Organisms can have their cells transformed

with a gene coding for a useful protein, such as an enzyme, so that they will overexpress the

desired protein

Mass quantities of the protein can then be manufactured by growing the transformed organism

in bioreactor equipment using industrial fermentation, and then purifying the protein

(Boundless, 2020).

Industrial applications include transforming microorganisms such as bacteria or yeast, or insect

mammalian cells with a gene coding for a useful protein (Boundless, 2020).

How’s it used in cheese making?

Anticipating a crisis of supply and demand, researchers turned to a then-unprecedented

technology in food science: genetically modified organisms (Levi G., 2015).

The result was a genetically altered bacterium that produced cheesemaking enzymes in a

beaker, instead of an animal's stomach (or rennet) that is used to clot milk they use other milk-

clotting enzymes that were genetically engineered. This revolutionized the cheese industry (Levi

G., 2015).

What is the function of your protein of interest?

Bromelain is the term given to a group of sulfhydryl proteolytic enzymes that contains cysteine

proteinases, one of the main groups of peptide-bond hydrolases, which is essential in breaking

down proteins and digesting food. This enzyme possesses anti-inflammatory properties that can

prevent postsurgical inflammation and trauma. It has the ability to relieve pain and increase

functionality for arthritic patients who suffers from cases of osteoarthritis and knee injury,

breaks down mucus and used as treatment for bronchitis and sinusitis, removes dead and

infected tissues that can stimulate growth of bacteria, prevents blood clotting

(thrombophlebitis) and growth of tumor (apoptotic cell death), contains anticancer properties,

enhances absorption of antibiotics, and counters diarrhea and effects of intestinal pathogens

(Vibrio Cholera & Escherichia Coli). Bromelain supplements also interfere with platelet

aggregation that reduces cardiovascular disorders.

 
However, when administering Bromelain to ease pain, it must not be consumed with actual

food to prevent it from acting as a digestive enzyme and patients’ varying tolerance to high

dosage of Bromelain may cause side effects such as diarrhea, nausea, dry mouth, flatulence,

allergic reaction, and skin irritations (S. Wilson Wijeratnam, 2016; A., Zengion, MSAOM, E.,

Yarnell, 2001).

In the food industry, bromelain has been providing aid for baking processes, tenderizing of

meat, beer clarification, food supplements, and prohibits enzymatic browning of apple juice.

Peeling effects in cosmetic industries and preparation of skin for tanning, softening and bating

in leather industries were also made possible by this enzyme. For leather jacket’s insoluble

protein, Bromelain performs as hydrolyzing agent for the release of antimicrobial peptides and

it is also used to improve the dyeing qualities and partially solubilizing protein fibers in silk and

wool (Bala et al.,2013).

What is the origin of your protein of interest? What (If any) modification were made to allow

for the production of this protein by industrial fermentation?

Bromelain is extracted in the stems, pulp, and even the inedible parts of pineapple (Ananas

comosus), a member of the Bromeliaceae Family, and derived from a mixture of proteases that

includes the following components: phosphatase, glucosidases, peroxidases, cellulases and

glycoproteins. The major protease found in the pineapple stem is the Stem Bromelain

(E.C.3.4.22.32) wherein the presence of other minor thiol endopeptidases—ananain and

comosain were observed. It is activated by Cysteine, an amino acid containing sulfur which
plays a vital role in protein synthesis, detoxification, and metabolic functions. The enzyme is

deactivated through metals like mercury and silver, and L-trans-epoxysuccinyl-leucylamido (4-

guanidino) butane. Most of the commercially available products in the market are obtained

from the Stem Bromelain. On the other hand, Fruit Bromelain (E.C. 3.4.22.33) is a moderately

present acidic protein that is found in the pineapple juice. It performs higher proteolytic activity

than Stem Bromelain and has broader distinction for substrates (S. Wilson Wijeratnam, 2016;

R., Pavan, S., Jain, Shraddha, A., Kumar, 2012)

How is your protein of interest produced? How is it utilized post-production?

Besides the clinical applications of Bromelain, it also has a lot of benefits in many industries to

date. Because of that, scientists are conducting their research on ways of extracting Bromelain

at its purest form while at the same time, cutting the cost of production. (Arshad et al. 2014)

Bromelain is highly concentrated on the stem of a pineapple, thus making it the best source for

extraction. (Tochi et al. 2008) The commercially available Bromelain is extracted by means of

ultrafiltration, centrifugation, lyophilization (Corzo et al. 2011) and two-step Fast Protein Liquid

Chromatography (FPLC) (Harrach et al. 1998). After the extraction process, the crude mixture

goes through several process of purification to remove unwanted impurities that may impede a

certain activity of the enzyme. (Illanes 2008) Several techniques of purification was found to be

ineffective due to their low purification potential so the extraction strategy must be selective

depending on the desired product.

For Bromelain to be utilized, it is recovered first from the Stem Bromelain through the use of

ethanol precipitation at low temperature. Concentration of ethanol must be ranging from 30%

to 70% to achieve a 2.28 purification factor which increases recovery of enzymatic activity to

98% (C., Bhattacharjee et al., 2015)

What are the scientific, social, and ethical implications of using genetic engineering methods

to manufacture proteins such as your protein of interest?

The main and probably most implications of genetic engineering are for science: for the

betterment of medicine and human health. This includes production of insulin for diabetic

patients, human growth hormones, antibodies, vaccines and several more others. This also

boost the researches and studies regarding genetic engineering to produce protein.

Socially, this may give positive implications to help our society. Manufactured proteins help us

to improve our healthcare and also food. Genetic engineering provides crops and farms

resistance to the factors that may cause it to decay and give these crops proteins to improve

the quality of the fruits and vegetables.

But as every other ‘too good things’, it has its own side and bad effect socially and ethically. As

Patra (2015) stated, “As more human genes are being used in non-human organisms to create

new forms of life that are genetically partly human, new ethical questions arise. For instance,

what percentage of human genes does an organism have to contain before it is considered
human. Human genes are now being inserted into tomatoes and peppers to make them grow

faster. This suggests that one can now be a vegetarian and a cannibal at the same time.”

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