Biotechnology project

protein engineering
Protein engineering is the design of new enzymes or proteins with
new or desirable functions. It is based on the use of recombinant
DNA technology to change amino acid sequences. the prospects
for protein engineering, such as X-ray crystallography, chemical
DNA synthesis, computer modelling of protein structure and folding
were discussed and the combination of crystal structure and protein
chemistry information with artificial gene synthesis was emphasized
as a powerful approach to obtain proteins with desirable properties.
Today, owing to the development in recombinant DNA technology
and high-throughput screening techniques, protein engineering
methods and applications are becoming increasingly important and
widespread.
It involves improvements of existing proteins such as enzymes
antibodies etc. And to create proteins which can resist
denaturation.
Protein engineers who are actually genetic engineers use
recombinant DNA technology to alter a particular nucleoside or
triplet that is codon in the DNA of a cell in this way it is hoped that
resulting dna codes for the different or new amino acids in the
desired location in the protein produced by that cell.
A study of 3 dimensional structure of the protein is a preliminary
step in protein engineering. A 3D structure of protein is produced
from the data generated from X-ray crystallography and NMR
(nuclear magnetic resonance) process by protein modelling.
Engineered proteins are used in drug development, food process-
ing, industrial manufacturing etc.
The objectives of protein engineering are as follows-
1. to produce enzymes in large quantities.
2. to produce proteins that are superior to natural ones.
3. numerous attemts have been made on different proteins and
enzymes inorder to improve their different properties of-
 Thermal stability
 pH stability
 solvent tolerance
 solubility
 catalytic potency
4. the engineering of proteins is even done to increase the stability
when exposed to
 harsh conditions
 elevated temperatures
 organic solvents
 reactive chemicals
5. to create a superior enzyme to catalyze the production of high
value specific chemicals.
6. Engineering may increase the efficiency of a particular enzyme
or protein therefore efforts have been made to alter the properties
of enzymes or proteins.
Rational design
In rational protein design, the scientist uses detailed knowledge of
the structure and function of the protein to make desired changes.
In general, this has the advantage of being inexpensive and
technically easy, since site-directed mutagenesis techniques are
well-developed. However, its major drawback is that detailed
structural knowledge of a protein is often unavailable, and, even
when it is available, it can be extremely difficult to predict the
effects of various mutations.
Computational protein design algorithms seek to identify novel
amino acid sequences that are low in energy when folded to the
pre-specified target structure. While the sequence-conformation
space that needs to be searched is large, the most challenging
requirement for computational protein design is a fast, yet accurate,
energy function that can distinguish optimal sequences from similar
suboptimal ones.

Directed evolution
In directed evolution, random mutagenesis is applied to a protein,
and a selection regime is used to pick out variants that have the
desired qualities. Further rounds of mutation and selection are then
applied. This method mimics natural evolution and, in general,
produces superior results to rational design. An additional
technique known as DNA shuffling mixes and matches pieces of
successful variants in order to produce better results. This process
mimics the recombination that occurs naturally during sexual
reproduction. The advantage of directed evolution is that it requires
no prior structural knowledge of a protein, nor is it necessary to be
able to predict what effect a given mutation will have. Indeed, the
results of directed evolution experiments are often surprising in that
desired changes are often caused by mutations that were not
expected to have that effect. The drawback is that they require
high-throughput, which is not feasible for all proteins. Large
amounts of recombinant DNA must be mutated and the products
screened for desired qualities. The sheer number of variants often
requires expensive robotic equipment to automate the process.
Furthermore, not all desired activities can be easily screened for.

APPLICATIONS OF PROTEIN
ENGINEERING
Applications in food industry
Early reports on the importance of protein engineering methods to
design new enzymes for enzyme biotechnological industries date
back to 1993. Particularly, the enzymes used in food industry were
emphasized as an important group of enzymes, the Protein
Engineering Methods and Applications industrially important
properties of which could be further improved by protein enginee-
ring.
An important application area of protein engineering regarding food
industry is the wheat gluten proteins. Wildtype
and mutant wheat gluten proteins were produced to compare them
to each other forprotein structure-function studies. Generally, E.coli
expression systems were suggested assuitable systems for many
applications, because of their availability, rapid and easy use,
aswell as high expression levels . Food industry makes use of
avariety of food-processing enzymes, such as amylases and
lipases, the properties of whichare improved using recombinant
DNA technology and protein engineering. The deletion ofnative
genes encoding extracellular proteases, for example, increased
enzyme productionyields of microbial hosts. In fungi, for example,
the production of toxic secondary metabolites has been reduced to
improve their productivity as enzyme-producing hosts.

Medical application
Medical applications of protein engineering are also diverse. The
use of protein engineering for cancer treatment studies is a major
area of interest. Pretargeted radioimmunotherapy has been
discussed as a potential cancer treatment. By pretargeting,
radiation toxicity is minimized by separating the rapidly cleared
radionuclide and the long-circulating antibody. Advances in protein
engineering and recombinant DNA technology were expected to
increase the use of pretargeted radioimmunotherapy . The use of
novel antibodies as anticancer agents is also an important field of
application, where the ability of antibodies to select antigens
specifically and with high affinity is exploited, and protein
engineering methods are used to modify antibodies to target cancer
cells for clinical applications . Recently, the term “modular protein
engineering” has been introduced for emerging cancer therapies.
Treatment strategies based on targeted nanoconjugates to be
specifically directed against target cells are becoming increasingly
important. Additionally, multifunctional and smart drug vehicles can
be produced at the nanoscale, by protein engineering. These
strategies could be combined to identify and select targets for
protein-based drug delivery . Protein engineering applications for
therapeutic protein production is an important area, particularly for
medicine. In 1996, recombinant protein production for therapeutic
purposes was reviewed. It was stated that protein engineering
resulted in a second generation of therapeutic protein products with
application-specific properties obtained by mutation, deletion of
fusion. The third generation of such products were mentioned as
“gene therapy” protein products to be produced by the patients,
upon gene transfer . Other studies on therapeutic protein
production include single-chain Fv designs for protein, cell and
gene therapy . DNA shuffling and recursive genetic recombination
studies to improve therapeutic proteins development of secreted
proteins such as insulin, interferon, erythropoietin as
biotherapeutics agents combinatorial protein biochemistry for
therapeutics and proteomics meganucleases and DNA double-
strand breakinduced recombination for gene therapy, the use of
protein cationization techniques for future drug discovery and
development protein display scaffolds for protein engineering of
new therapeutics and polymer-based therapeutics for drug delivery
and tissue regeneration.
Protein engineering applications with antibodies are also diverse.
Owing to advances in recombinant DNA technology, “antibody
engineering” is possible. Improvements such as minimal
recognition units and antigenized antibodies were described.
Combinational approaches such as bacteriophage display libraries
have been introduced as a strong alternative to hybridoma
technology for antibody production with desired antigen binding
characteristics. Studies on genetic manipulation of mouse
monoclonals for producing humanized antibodies and
bacteriophage display libraries for Ig repertoires have been
reported . Phage display has become a powerful technique in
protein engineering, immunology, oncology, etc. Phage display of
antibody fragments, particularly the production of artificial epitopes
by phage antibodies is an important application. “Antibody
modeling” studies to engineer antibody-like molecules and increase
their stability and specificity are also common, particularly for
humanization of antibodies of animal origin. Recently, the use of
antibodies as vectors for molecular imaging has become popular.
Pharmacokinetic properties of antibodies have been improved by
protein engineering and antibody variants of different size and
antigen binding sites have been produced for the ultimate use as
imaging probes specific to target tissues. A variety of examples
include antibody fragments which have been conjugated to
bioluminescence, fluorescence, quantum dots for optical imaging,
as well as iron oxide nanoparticles for magnetic resonance
imaging. It is obvious that molecular imaging tools based on
antibodies will find more applications in the future regarding
diagnosis and treatment of cancer and other complex diseases.
Improving laundry detergent Subtilisin
Subtilisin is a protease produced by bacteria that can digest a
broad range of proteins that commonly soil clothing, The enzymatic
activity of subtilisin is contributed by a catalytic triad, i.e., Ser221,
His64 and Asp32 similar to chymotrypsin. Replacement of all three
residues with alanine either singly or in combination results in
significant loss of activity. Subtilisin represents the largest industrial
market for any enzyme.
The native enzyme subtilisin is easily inactivated by bleach (up to
90%). Careful studies showed that this inactivation was due to
oxidation of the amino acid residue Methionine222 in the protein
molecule. Using site-directed mutagenesis of the subtilisin gene in
E.coli, this methionine was substituted by a variety of other amino
acids and the enzyme activity measured in the presence of bleach .
It was observed that substitution of Met222 with Ala222 was the
best in terms of activity and stability. Nowadays, many laundry
detergents contain cloned, genetically engineered or recombinant
subtilisin.

The modification of natural enzymes and proteins by protein

engineering is an increasingly important scientific field. The well-
known methods of rational design and directed evolution, as well as
new techniques will enable efficient and easy modification of
proteins. New technologies such as computational design, catalytic
antibodies and mRNA display would be crucial for de novo
engineering of enzymes and also for new areas of protein engine-
ering. Protein engineering applications cover a broad range,
including biocatalysis for food and industry, as well as medical,
environmental and nanobiotechnological applications. With
advances in recombinant DNA technology tools, “omics” technolo-
gies and high-throughput screening facilities, improved methods for
protein engineering will be available, which would enable easy
modification or improvement of more proteins/ enzymes for further
specific applications.