1. Improvement in Subtilisin – A Biological Laundry Detergent :
Subtilisin is an alkaline protease of 27.5 kilo Dalton which is isolated from a bacterium, Bacillus subtilis. Subtilisin is most commonly used by detergent industry in 95% of washing as a stain- remover detergent formulation. The protein stains are removed more effectively and at lower temperature than are usually required for laundry processing.
The ideal requirements for such enzymes are: (i) stability up to 70°C and within the pW range of 8-11, (ii) resistance to non-ionic detergents and oxidising reagents such as H2O2, and (iii) the absence of requirement of metal ions. Substilisin acts upon stains by catalytic triad (like chymotrypsin) i.e. Asp32, His64 and Ser21.
Significantly, enzyme activity is lost if all the three amino acids are replaced either singly or in combinations. By adding bleach about 90% activity of subtilisin is lost due to oxidation of an amino acid methionine present at position 222 (Met222) of the polypeptide chain.
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The methionine was substituted by either alanine or cysteine through site-directed mutagenesis in subtilisin gene. The Ala222 substituted enzyme activity of engineered subtilisin is measured in the presence of bleach (Table 2.2). The alanine222 substituted enzyme showed 53% activity as compared to wild type one.
The best stability and activity of the engineered subtilisin was noted where Met222 was replaced by alanine. At present recombinant subtilisin is used in laundry detergent by many detergent industries.
2. Recombinant Vaccines :
Earlier vaccine were prepared by inactivating the bacteria/viruses or their surface proteins. Such bacterial or viral preparations have been used to generate immunity against specific bacterial or viral diseases.
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It has been noted that patients often develop fever who were administered the vaccines. It is true that proteins stimulate immune system and cause to secrete specific antibodies.
Such specific amino acid sequences in the protein that stimulate immune response are called epitopes. Based on selected epitomes recombinant vaccines may be produced on commercial level which can prove more effective and safer than the conventional vaccines.
Working on these lines, a recombinant Hepatitis B vaccine was produced by cloning the synthetic gene (for the surface antigen of the virus) in yeast cells.
This gene expressed well in yeast cells and produced 22 nm particles of hepatitis B virus (HBV) surface antigen (as produced in patients) infected with hepatitis B virus.
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The recombinant vaccine has high immunogenecity. This product has been marketed as a vaccine for protection against HBV infection. Similarly, recombinant vaccines for foot and mouth disease (FMD) virus have been prepared.
3. Protein Engineering in Enzymes :
There are many enzymes where protein-engineering has been done. Two enzymes viz., triosephosphate isomerase and P-lactamase have been discussed.
(a) Triosephosphate Isomerase:
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Proteins exposed at high temperature releases ammonia due to deamination of asparagine and glutamine residues. Hence, enzyme activity is lost due to localised changes in folding of protein.
For example, triosephosphate isomerase is a yeast (Saccharomyces cerevisiae) enzyme which has two similar sub-units. Each sub-unit consists of two asparagine residues present at position 14 and 78 and these provide thermostability to the enzyme. These as- Saccharomyces cerevisiae.
paragine residues were replaced by threonine or isoleucine using a site-directed mutagenesis. The engineered enzyme was more thermostable and resistant to proteolysis than the wild type enzyme.
(b) Beta-lactamase:
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The ß-lactamase is secreted by bacteria to degrade penicillin. This enzyme functions in periplasmic space of bacterial cell and inactivates the (3-lactam ring of penicillin. Consequently it is transported across the plasma membrane.
During transport a signal polypeptide of 23 amino acids are broken. Beside this polypeptide, transport and processing depend on other polypeptides also.
An active site consisting of amino acid serine has been detected. Activity of this enzyme can be reduced after replacing serine by cysteine.
4. Protein Design :
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The main objective of protein-engineering is to design a chain of amino acids which may fold to attain the desired structure so that desired function can be done.
On the basis of known protein sequences and structure it is easy to design a chain of amino acids for specific structure and function than to predict the specific sequence.
Considering these facts a globular water soluble protein without any specific function was designed. It attained definite secondary and tertiary structure.
This protein consists of four a-helix which fold to form a compact bundle. This motif is common in proteins. Hydrophobic amino acids are to the interior and hydrophilic amino acids to the exterior. Four 16-residue long sequence of the four helix region was as below:
This four 16-residue helix sequence was connected by three short loop regions having amino acids: Pro-Arg-Arg-. The corresponding DNA sequence was synthesised and incorporated into E. coli by following recombinant DNA technology. It expressed in expression vector system.
The second example is the design of an eye lens protein Crystanova. Gene encoding this protein was synthesised and incorporated into E. coli which expressed crystanova.
5. Improvement in Nutritional Quality of Seeds :
The major source of protein of our diet is the seeds and cereal grains. During the development of seeds, the ‘seed storage protein’ are synthesised and accumulated. When such seeds germinate or being conserved by humans its reserved proteins act as source of amino acids.
However, if some amino acids lack in these seeds or grains, these cannot be used as balance diet. Therefore, those essential amino acids are supplemented to the diets procuring from the other sources.
There are 20 amino acids; all are not essential. The essential amino acids are given in.
Out of the proteins found in nature, all are not consumable. Because some are toxic or allergenic in nature. Therefore, such proteins should be consumed which are nutritious and safe. The highly nutritious food materials which we consume are easily contaminated by the microorganisms.
During growth and multiplication the microorganisms secrete toxins like mycotoxins by fungi (such as aflatoxins, ochratoxin, rubratoxin, ergot, etc.). These toxins cause several health hazards in animals and human too.
However, there are several measures to estimate the nutritional value of food protein which meets the nutritional requirement of an individual. These parameters of protein value are essential amino acid profile (EAP), biological value (BV) or net protein efficiency ratio (PER).
(a) Essential Amino Acid Profile:
The amino acids which are taken up from external food are called essential amino acids. Our body cannot synthesise them. EAP of different protein sources is given in.
Amount of branched chain amino acids (BCAAs) i.e. lie, Leu, Tys, Thr and Val is highest in whey as compared to the others. BCAAs need to be present in muscle cells so that protein synthesis should be promoted. In the presence of BCAA, bioavailability of complex carbohydrates is increased.
When a person performs exercises, BCAAs are released from skeletal muscle. Its carbon part is utilised for energy production and nitrogen part is converted into alanine. Alanine is transported into liver and takes part in energy production.
Therefore, BCAA are the good source of energy for athletes during, before and after exercises and protecting their biomass from breakdown of muscles.
(b) Biological Value (BV):
BV is the percentage of protein nitrogen retained in body after consuming the known amount of protein nitrogen. Because there is simultaneous loss of endogenous nitrogen through excretion of urine. Whey proteins have the highest BV as compared to rice, wheat, soyabean and egg proteins. BV is expressed by the following formula:
(c) Protein Efficiency Ratio (PER):
PER is a growth parameter which is expressed in terms of weight gain by an animal or individual after feeding/consuming 1 g of protein (as compared to reference protein). The PER of various proteins in increasing order is given below:
Wheat protein > rice protein > soya protein > casein > milk protein > whey protein
Most of the grains/seeds which we eat are deficient in storage proteins and desirable amino acids. Therefore, today’s scientists are making effort to engineer new genes which can synthesis storage proteins containing desirable amino acids.
For the increase of nutritional value of maize scientists have already made attempts to engineer genes of seen storage protein. Beside, fully new genes for said purpose can also be inserted.
(d) Digestibility (D):
An individual is given known amount of protein food. After consuming protein food nitrogen content in faeces is estimated. The percentage of total nitrogen consumed after absorption through alimentary tract is calculated by using the following formula:
The term ‘proteome’ is used to describe the total set of proteins expressed from the transcriptome of a cell. But the term proteome has been devised to describe the proteins specified by genome of an organism.
Proteome is a wide term which includes all the variants of a single gene product that are produced from alternative splicing of transcribed RNA and from post-transcriptional modification of a single protein product.
Proteomics is the direct outcome of advancement made for nucleotide sequencing of different genomes in large scale. This helps to identify various proteins. Generation of information about protein is necessary.
Because, protein governs the phenotypic characters of the cells. Merely genome study cannot provide the understanding of mechanism of disease development and various developmental changes occurring in organisms including humans.
Moreover, target drugs for many kinds of diseases can be prepared only after understanding the protein modification and protein functions. There are many areas of modern proteomics such as protein expression, protein structure, protein localisation, protein-protein interaction, etc.
About one third of gene sequences of organisms (of which genome sequence is known) do not carry out any function. The structural genomic projects can be assisted significantly only identifying the proteins completely.
One of the objectives of structural genomics is to prepare 3D structure of all proteins expressed by the genome of the cells. Based on the protein structures possible functions may be assigned to respective proteins.
A. Relation between Gene and Protein
In eukaryotes transcription occurs inside the nucleus and translation in cytoplasm. DNA is transcribed under transcriptional regulation into pre-mRNAs by RNA polymerase II.
Inside the nucleus, pre-mRNAs undergo various post-transcriptional modifications to increase their stability such as capping at 5′-end, polyadenylation (i.e. addition of poly (A) at 3-OH end) and mRNA addicting.
The introns are spliced out by splisosomes through a process called splicing. Then the mature mRNAs are transported from nucleus to cytoplasm for translation into protein on the ribosomes.
Translational regulation of protein occurs in cytoplasm. Then protein undergoes post-translational modifications to about 200 types.
B. Types of Proteomics :
There are many types of proteomics as shown in Fig. 2.10, but expression proteomics, structural proteomics and functional proteomics have been dealt herewith.
1. Expression Proteomics :
Expression proteomics is the quantitative study of protein expression between the samples which differ by some variables. A comparative study of the whole proteome between the samples can be done using this approach.
For example, a patient suffering from mouth cancer develops a small tumour. Thus tumour of cancer patient and similar tissues from a normal person may be taken out and analysed for protein expression following different routes.
Using techniques of high resolution protein separation and identification (e.g. two-dimensional gel electrophoresis, isoelectric focusing, MALDf mass spectrometry, microarray technique, etc.) under-expressed or over-expressed proteins in cancer patient and normal ones can be characterised and identified.
An understanding about the formation of such tumour could be developed on the basis of proteins identified and compared between the two individuals.
2. Structural Proteomics :
The structural proteomics deals with the study of structure and nature of protein complexes present in a particular cell organelle. To fulfill this objective specific sub-cellular organelles or all protein complexes are isolated.
All proteins present in these complexes are identified and protein-protein interactions occurring between them are characterised.
It shows 3D shape of ribonuclease A. These studies lend support to assemble information about the structural topography of the cells and clues how certain proteins got expressed and gave unique characteristics to the cells.
3. Functional Proteomics :
Functional proteomics is a broad term which embraces all proteomics approaches related to devising its functions. It is defined as the use of proteomics methods for analysis of properties of molecular networks formed in a living cell. In this study molecules are identified which take part in such networks.
Recently, some novel proteins have been discovered which transport the important molecules from nucleus to cytoplasm and cytoplasm to nucleus. This functional proteomics is rather a complex process where function of a molecule is found out in the molecular networks.