Protein Production by Genetic Engineering

Nowadays, proteins are used in many fields such as food production, vaccine, therapeutics, diagnostic tools, cosmetics, feed additives, textile and detergent industries. For these applications proteins have to be probably produced in large scales. Isolating proteins simply from their natural sources cannot fulfill this protein demand. So how these proteins are produces? Here comes the application of genetic engineering to the protein production. Genetic engineering is the artificial modification or recombination of DNA in order to modify an organism. Recombinant DNA technology is a popular technique used in genetic engineering. It is the joining of DNA fragments of two different species and producing a new genetic combination which giving an important output. This can be used to produce proteins in large scales. Proteins which are produced by recombinant DNA technology are called as the recombinant proteins.

Protein synthesis occurs through two main steps as transcription and translation. During transcription protein coding genes convert in to mRNA which carry the information of amino acids of the protein. This is done by RNA polymerase enzyme. mRNA is produces in the nucleus of the cell and go through the nuclear pores to the cytoplasm. Translation of the mRNA in to the protein take place in the ribosomes of the cell. It reads the message in the mRNA and determine the amino acid sequence. Particular amino acids are carried to the ribosome by tRNA. Amino acids combine each other with peptide bonds according to the order of the mRNA and produce the polypeptide chain. Then it folds to form a functional protein and undergo post-translational modifications. It is decided according to the type of the protein. After that they are localized to particular places where their action is needed. This natural process can be used to produce proteins commercially in large scales. First of all, we have to create our recombinant construct. For that some steps have to be followed.

1. Isolating the gene of interest and amplify the gene copies by PCR
2. Selecting a suitable vector
3. Ligation of gene fragment to the vector
4. Transformation of vectors to the host organism
5. Obtaining the recombinant product

Figure 1. Basic steps in gene cloning (T.A Brown; Gene cloning and DNA analysis: an introduction, 6th ed; John Wiley & Sons, Ltd., West Sussex, U.K, 2010, p 5)

First the gene of interest which code for the particular protein have to be isolated. As an example if we want to produce human insulin, we have to isolate the human insulin gene. Isolation of genes is a complex process with particular experimental conditions. Fragments should have complementary cutting sites/ restriction sites to the vector. Cutting of DNA fragments is done by restriction enzymes. PCR can be used to get multiple copies of the gene. Then it should be inserted in to the multiple cloning site of the vector to produce the recombinant molecule. There are two main types of vectors as cloning vectors and expression vectors. Vector selection should be done according to the process and the result that we expect. Here as we need to express the protein in large scale an expression vector should be used. It should have a strong promoter to get a high yield. The process of inserting the foreign DNA in to the vector is called as ligation, which is done by DNA ligase enzyme. Vector transport the DNA fragment in to the host cell. Introducing the recombinant DNA molecule to the host cell is knows as transformation. Before the transformation competent host cells should be prepared using chemical or physical treatments. Under normal conditions cells do not allow foreign genes to enter to the cells. Competent cell preparation makes the host cells permeable to the foreign DNA. Recombinant DNA can be introduced to the host cells by an electric or a heat shock. All the host cells do not have an equal efficiency at DNA uptake. There can be some cells without the vector that we introduced. Cells which carry the vector are called as transformed cells. Those cells should be separated for further process. For that characteristics of the vector can be used. Vector should have an antibiotic resistance which cannot be seen in the host cells. After the transformation host cells should be grown on an agar medium with that antibiotic. Host cells with the vector (transformers) able to form colonies in the medium. Non-transformers cannot produce colonies as they do not have the resistant gene, they are killed by the action of the antibiotic. Most vectors carry at least one gene that confers antibiotic resistance to the host cells. Thus transformers and non-transformers can be easily distinguished. Vector autonomously replicate inside the host cell by producing many identical copies. When the host cell divides, copies of the vector pass to the progeny. After many cell divisions a colony of identical host cells with one or more vectors can be obtain. After the process recombinants should be selected which having the ability to produce our interesting protein. Even the all transformers carry the vector, there can be self-ligated vectors which do not carry the foreign DNA. They are non-recombinants and do not have the ability to synthesize our interesting protein. Recombinants can be distinguished by the blue-white screening. Normally vectors carry lacZ gene, which code for part of the β-galactosidase enzyme. Foreign DNA insertion inactivates this gene. Recombinants can be identified by the inability to synthesize β-galactosidase. It is an enzyme which involve in lactose breakdown. Transformers should be introduced to an agar medium with X-gal (a lactose analogue) and IPTG (inducer for the enzyme). Cells which produce the enzyme breakdown X-gal in to blue color product. Because of that non-recombinants produce blue color colonies in the medium. Recombinants produce white color colonies as they cannot breakdown X-gal. The expression of the protein can be detected using western blotting and expressed protein can be purified by chromatographic techniques. It will be easy to purify if the protein can be secreted outside the host cell. There are many factors and experimental conditions that should be considered within this whole process.

There are many difficulties and challenges associated with this protein production which can be divided in to two categories.

1. Problems associated with the sequence of the foreign gene
2. Problems due to the limitations of the host for recombinant protein synthesis.

The nucleotide sequence can prevent the efficient expression of the cloned gene. If the foreign gene contains introns, it will unable to remove from the transcript when having a bacterial cell as the host. Because they don’t have a mechanism to remove it. This can be prevented by using cDNA prepared from mRNA. And also the foreign gene might be containing sequences that can act as termination signals in the host. In vitro mutagenesis can be used to change the possible terminators. Sometimes the host will be unable to fold the recombinant protein correctly. Because they may not synthesize the disulphide bonds present in the protein. If the protein does not have its tertiary structure it become insoluble and forms inclusion bodies within the bacterium. Protein can be isolated from the inclusion body, but it will be unable to convert in to the correct structure inside a test tube.  So the protein become inactive. This can be prevented by using a special host strain which suitable to the process. And also the host might degrade the recombinant protein. This can be reduced by using a mutant host strain that do not have the protease responsible for the protein degradation. When selecting a host all these factors should be concern for a successful result. Many recombinant proteins have been produced in host cells such as bacteria, yeast, mammalian, insects and plant. Human proteins have been successfully synthesized using genetic engineering. And they are using for many treatments in the medical field. Farm animals like cows, sheep and pigs have successfully cloned genes to the promoters in the mammary tissues. So the recombinant protein can be secreted in the milk. Transgenic plants provide relatively cheap and mass production of recombinant protein. And also there are some ethical concerns to be considered in transgenic animal and plant production. Thus genetic engineering has provided a new countenance to the protein production.

Table1; Some human proteins which are synthesized by genetic engineering                                       (T.A Brown; Gene cloning and DNA analysis: an introduction, 6^th ed; John Wiley & Sons, Ltd., West Sussex, U.K, 2010, p 251)

Prasadika samarawickrama
B.Sc. (Special) Degree in Biochemistry and Molecular Biology
Faculty of science
University of Colombo

References :
1. T.A Brown; Gene cloning and DNA analysis: an introduction, 6th ed; John Wiley & Sons, Ltd., West Sussex, U.K, 20102. https://www.cusabio.com/c-20679.html (accessed on 10/06/2020)
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