Introduction to Protein Engineering

Introduction to Protein
Engineering
6 June 2021 Abhijit Debnath BP605T and Biotech Unit-1 1
CO1.1
Noida Institute of Engineering and Technology (Pharmacy
Institute) Greater Noida

Introduction to
Protein
Engineering
 Human organ system and protein
 Basics of protein & amino acids
 Protein manufacturing in biological system
 Protein & peptides
 Levels of protein structure
 Body composition and protein
 Functions of proteins
 DNA to protein in nutshell
 Protein engineering
 Objectives/ need of protein engineering
 Rationale of protein engineering
 Factors influencing in engineering
 Challenges in protein engineering
 Protein engineering methods
 Applications/Success Stories
CO1.1
Noida Institute of Engineering and Technology
(Pharmacy Institute) Greater Noida

 Our Human Body having 11 Systems
Infrastructure Systems Regulation Systems Energy Systems
 All different Systems are made up of several types of Tissues
 All different type of Tissues are made up of different types of Cells
NM_000041
HUMAN ORGAN SYSTEM AND PROTEIN (CO1.1)

PROTEIN ENGINEERING (CO1.1)

Nucleotides are the smallest
building blocks of DNA. There are
four nucleotides (A, G, T, C) which
arrange in pairs to form the long
double strands typical of DNA
molecules.

Gene is a segment of DNA which
codes for the amino acid
sequence of a particular protein. A
gene is therefore composed of
many pairs of nucleotides.
Eg. APOE e4 Gene appears to
increase the risk of Alzheimer
Disease.

Chromosome is a long strand of
DNA which is coiled up with
various proteins.
A chromosome contains many
genes.

Genome is all the DNA of a
particular organism. All of an
organism's chromosomes
compose the organism's genome.

Facts-
There are
 37.2 trillion cells in Human Body!
 23 chromosome pairs
 with a total of about 3 billion DNA base pairs
 About 25,000 genes are present in Human Genome;
 about 20,000 of these genes are protein-coding genes!
 Humans make at least 20,000 proteins!
 Amino acids are the building blocks these Proteins!
 Total 21 Amino acids

BASICS OF PROTEIN & AMINO ACIDS (CO1.1)

PROTEIN MANUFACTURING IN BIOLOGICAL SYSTEM (CO1.1)

6 June 2021 Abhijit Debnath BP605T and Biotech Unit-1
PROTEIN MANUFACTURING IN BIOLOGICAL SYSTEM (CO1.1)
13

PROTEIN MANUFACTURING IN BIOLOGICAL SYSTEM(CO1.1)

 Proteins: more than 100 amino
acid monomers
 Peptide: less than 100 amino
acid monomers
PROTEIN & PEPTIDES (CO1.1)

LEVELS OF PROTEIN STRUCTURE (CO1.1)

BODY COMPOSITION AND PROTEIN (CO1.1)

The estimated gross molecular contents of a typical 20-micrometre human cell is as
follows
BODY COMPOSITION AND PROTEIN (CO1.1)

1. Enzymes- Metabolism
2. Structural- Collagen and Keratin
3. Cell Recognition- proteins on cellular surface.
4. Regulation of Gene Expression-
Gene Repressors or Enhancers.
5. Défense- Antibodies.
FUNCTIONS OF PROTEINS (CO1.1)

DNA TO PROTEIN IN NUTSHELL (CO1.1)

 Protein engineering can be defined as the modification of protein
structure with recombinant DNA technology or chemical treatment
to get a desirable function for better use in medicine, industry and
agriculture.
 Protein engineering the process of developing useful valuable
proteins.
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OBJECTIVES/ NEED OF PROTEIN ENGINEERING (CO1.1)
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 The objectives of protein engineering is as follows –
 To create a superior enzyme to catalyze the production of high value specific chemicals.
 To produce enzyme in large quantities.
 To develop useful valuable proteins
 To get a desirable function for better use in
medicine, industry and agriculture.

 Elimination of allosteric Regulations
 Improved kinetic properties
 To produce biological compounds(include synthetic
peptide, storage protein, and synthetic drugs) superior to
natural one.

 Enhanced substrate and reaction specificity
 Increased thermostability.
 Alteration in optimum pH
 Solubility for use in organic solvents.
 Increased / decreased Optimal temperature
 To speed up the process( rate of reaction)
 Increase Protein/ Enzymes' Shelf Life
 To get high quality of Product Suitability face
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•Valdecoxib (Bextra)
•Pemoline (Cylert)
•Bromfenac (Duract)
•Levamisole (Ergamisol)
•Rofecoxib (Vioxx)
•Isotretinoin (Accutane)
•Sibutramine (Meridia)
•Terfenadine (Seldane)

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For industrial application an enzyme, should possess some characteristics in addition to those of enzymes in
cells. These characteristics are :-
 Enzyme should be robust with long life.
 Enzyme should be able to use the substrate supplied in the industry even it differs from that in the cell.
RATIONALE OF PROTEIN ENGINEERING (CO1.1)
30

 Enzyme should be able to work under conditions, e.g. extreme of pH, temperature and concentration of the
industry even if they differ from those in the cell.
 In view of above, the enzyme should be engineered to meet the altered needs. Therefore efforts have been
made to alter the properties of enzymes.

 These are some character that one might have to change in a predictable manner in protein engineering or enzyme
engineering to get the desired function

 Characteristics that one might have to change in a predictable manner in protein engineering or enzyme
engineering to get the desired function :-
 Kinetic properties of enzyme-turnover and Michaelis constant, Km.
 Thermo stability and the optimum temperature for the enzyme.
 Stability and activity of enzyme in nonaqueous solvents.
 Substrate and reaction specificity.
 Cofactor requirements
 Optimum PH.
FACTORS INFLUENCING IN ENGINEERING (CO1.1)

 Characteristics that one might have to change in a predictable manner in protein engineering or enzyme
engineering to get the desired function :-
 Molecular weight and subunit structure.
 Therefore for a particular class of enzymes, variation in nature may occur for each of the above
properties, so that one may like to combine all the optimum properties to the most efficient form of the
enzyme.
 For an e.g. glucose isomerases, which convert glucose into other isomers like fructose and are used to
make high fructose corn syrup vital for soft drink industries.
FACTORS INFLUENCING IN ENGINEERING (CO1.1)

 Uniport: 214,971,037 Sequences
 Protein Data Bank: 78229 structures
CHALLENGES IN PROTEIN ENGINEERING (CO1.1)
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 Issues with Protein Engineering :- Non availability of 3D Structure of Protein

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Protein Sequence 3D Structure ?

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 Protein X-ray crystallography
This technique used to obtain the three-dimensional structure of a particular
protein by x-ray diffraction, but it is not easy.

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Homology modeling
 MODELLER
 SwissModel
 BhageerathH+
Threading/fold recognition is
 IntFOLD
 RaptorX
Ab initio structure prediction is
 trRosetta
 I-TASSER
Critical Assessment of Techniques for Protein Structure
Prediction (CASP)
 CASP organizers had been posting on this website
sequences of unknown protein structures for
modeling.
 Independent mechanism for the assessment of
methods of protein structure modeling
 Computational Approaches:
Low accuracy !

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Critical Assessment of Techniques for Protein Structure Prediction (CASP) 12

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Rational design of Protein
1. Site directed mutagenesis
2. Protein Engineering by use of gene families
3. Protein Engineering through chemical modification
4. Directed evolution
5. Asexual methods
6. Sexual methods
7. In vitro non-homologous recombination methods
8. Exon shuffling
9. Semi-rational design
10. Screening and selection techniques
11. . De novo enzyme engineering:
12. Random mutagenesis
13. Focused mutagenesis
PROTEIN ENGINEERING APPROCHES (CO1.1)
41

Rational design:
 Rational designing of protein is the supreme regular way of protein engineering
 It is an effective approach when the structure and mechanism of the protein of interest are well-known.
 In many cases of protein engineering, however, there is limited amount of information on the structure and
mechanisms of the protein of interest.
 Thus, the use of “evolutionary methods” that involve “random mutagenesis and selection” for the desired
protein properties was introduced as an alternative approach.
 Application of random mutagenesis could be an effective method, particularly when there is limited
information on protein structure and mechanism.
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Rational design:
 The only requirement here is the availability of a suitable selection scheme that favors the desired protein
properties (Arnold, 1993).
 A simple and common technique for random mutagenesis is “saturation mutagenesis”.
 It involves the replacement of a single amino acid within a protein with each of the natural amino acids, and
provides all possible variations at that site.
 “Localized or region-specific random mutagenesis” is another technique which is a combination of rational
and random approaches of protein engineering.
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Rational design:
 It includes the simultaneous replacement of a few amino acid residues in a specific region, to obtain proteins
with new specificities.
 This technique also makes use of overlap extension, and the whole-plasmid, single round PCR mutagenesis,
as in the case of site-directed mutagenesis.
 However, the major difference here is that the codons for the selected amino acids are randomized, such
that a mixture of 64 different forward and 64 different reverse primers are used, based on a statistical
mixture of four bases and three nucleotides in a randomized codon (Antikainen & Martin, 2005).
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 Requirements:
1. Knowledge of sequence and preferable Structure (active site,....)
2. Understanding of mechanism (knowledge about structure - function relationship)
3. Identification of cofactors
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 Mutations Means:
46

In this Process Mutation is developed at specific site.
Step 1: In M13 Virus or bacteriophage has Single strand DNA (Plasmid)
Step 2: The Gene which is to be changed whose single stranded copy inserted in M13 Virus or Phase DNA
Step 3: Now this single stranded copy allowed to get double stranded
Step 4: Now in outer strand one nucleotide is substituted by another nucleotide.
Step 5: Now this mutated DNA allow to enter
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2. Protein Engineering by use of gene families
 This technique involves isolation of Gene from each species, and create hybride for eq-subtilisin (an enzyme used
detergent industry), genes from 26 species were mixed so we get 4 types of subtilisin enzyme with improved
qualities in different aspect.
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3. Protein Engineering through chemical modification
 In this method different chemical used as "Glutaraldehyde". It acts as protein cross-linker. It stabilizes the protein
in solutions
 By the Glutaraldehyde" different Proteine as haemoglobin, insulin Phosphofructokinase, Lactate dehydrojenase
have been stabilized
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 Rational techniques of protein engineering have limitations based on limited understanding of protein folding,
hence urging another approach to arise which is known as directed evolution.
 It is applied to a protein and a selection regime is used to select variants having desired traits.
 Further rounds of mutation and selection are then applied.
 This method mimics natural evolution and, in general, produces superior results to rational design.
 An added process, termed DNA shuffling, mixes and matches pieces of successful variants to produce better results.
 Such processes mimic the recombination that occurs naturally during sexual reproduction.
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 Advantages are 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.
 The results of directed evolution experiments are often surprising in that desired changes are often caused by mutations
that were not expected to have some effect.
 The drawback is that they require high-throughput screening, which is not feasible for all proteins.
 Large amounts of recombinant DNA must be mutated and the products screened for desired traits.
 The large number of variants often requires expensive robotic equipment to automate the process. Further, not all
desired activities can be screened for easily.
 Directed evolution methods can be broadly categorized into two strategies, asexual and sexual methods.
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5. Asexual Methods
 Asexual methods do not generate any cross links between parental genes.
 Single genes are used to create mutant libraries using various mutagenic techniques.
 These asexual methods can produce either random or focused mutagenesis.
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6. Sexual methods
 Sexual methods of directed evolution involve in vitro recombination which mimic natural in vivo recombination.
 Generally these techniques require high sequence homology between parental sequences.
 These techniques are often used to recombine two different parental genes, and these methods do create cross
overs between these genes.
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7. In vitro non-homologous recombination methods
 These methods are based upon the fact that proteins can exhibit similar structural identity while lacking sequence
homology.
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8. Exon shuffling
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8. Exon shuffling
 Exon shuffling is the combination of exons from different proteins by recombination events occurring at introns.
 Orthologous exon shuffling involves combining exons from orthologous genes from different species.
 Orthologous domain shuffling involves shuffling of entire protein domains from orthologous genes from different
species.
 Paralogous exon shuffling involves shuffling of exon from different genes from the same species.
 Paralogous domain shuffling involves shuffling of entire protein domains from paralogous proteins from the same
species.
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8. Exon shuffling
 Functional homolog shuffling involves shuffling of non-homologous domains which are functional related.
 All of these processes being with amplification of the desired exons from different genes using chimeric synthetic
oligonucleotides.
 This amplification products are then reassembled into full length genes using primer-less PCR.
 During these PCR cycles the fragments act as templates and primers.
 This results in chimeric full length genes, which are then subjected to screening.
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 Semi-rational design uses information about a proteins sequence, structure and function, in tandem with
predictive algorithms.
 Together these are used to identify target amino acid residues which are most likely to influence protein function.
 Mutations of these key amino acid residues create libraries of mutant proteins that are more likely to have
enhanced properties.
61

 Advances in semi-rational enzyme engineering and de novo enzyme design provide researchers with powerful and
effective new strategies to manipulate biocatalysts.
 Integration of sequence and structure based approaches in library design has proven to be a great guide for enzyme
redesign.
 Generally, current computational de novo and redesign methods do not compare to evolved variants in catalytic
performance.
 Although experimental optimization may be produced using directed evolution, further improvements in the accuracy
of structure predictions and greater catalytic ability will be achieved with improvements in design algorithms.
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10. Screening and selection techniques
 Once a protein has undergone directed evolution, ration design or semi-ration design, the libraries of mutant
proteins must be screened to determine which mutants show enhanced properties.
 Phage display methods are one option for screening proteins.
 This method involves the fusion of genes encoding the variant polypeptides with phage coat protein genes.
 Protein variants expressed on phage surfaces are selected by binding with immobilized targets in vitro.
 Phages with selected protein variants are then amplified in bacteria, followed by the identification of positive
clones by enzyme linked immunosorbent assay.
 These selected phages are then subjected to DNA sequencing.
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11. De novo enzyme engineering: De novo synthesis of enzymes reflects that enzyme are being synthesized from the
scratch and with respect to their reaction or substrate mechanism; these are not centred on their related parent
enzyme. The de novo synthesis can be done by employing
(i) In silico rational design;
(ii) Knowledge of a reaction mechanism and
(iii) mRNA display to search large protein libraries.
It is far much easier to search de novo proteins from larger libraries using mRNA display method as
compared to cell surface and phage display techniques, because the mRNA makes covalent bond with the protein
encoded by it and makes the direct amplification of desired protein simpler.
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12. Random mutagenesis:
 Directed evolution is a powerful technique for generating tailor-made enzymes for a wide range of
biocatalytic applications.
 Following the principles of natural evolution, iterative cycles of mutagenesis and screening or selection are
applied to modify protein properties, enhance catalytic activities or develop completely new protein catalysts for
non-natural chemical transformations. .
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13. Focused mutagenesis:
 Random mutagenesis can create great number of libraries, though they might not be rich in functional proteins.
 Many of them might have lethal mutations due to which protein folding may not take place or it might get deadly
functional.
 Alternatively, focused mutagenesis has been developed that involves producing mutations at specific sites of
proteins probably being a catalytic site or a functional region, therefore, yielding a library of functionally rich
proteins.
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PROTEIN ENGINEERING APPROCHES (CO1.1)
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APPLICATIONS/SUCCESS STORIES (CO1.1)`
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 Industrial applications:
• A wide-ranging enzymes are being employed in diverse industries like food, paper and leather, cosmetic,
pharmaceutical and chemical industry.
• Scientists have been noticed to begin protein engineering to produce new enzymes for biotechnological
industries from early 1990s.
• Predominantly, food industry expenditures a variety of enzymes like proteases, lipases, amylases etc. in
food processing.

71
 Applications referring to remediation of polluted environments oxygenases, laccases and peroxidases are
three major classes of enzymes, which have significant role in environmental applications for biodegradation
of organic and toxic pollutants.
• these enzymes face problems like enzyme denaturation by toxic compounds, inhibition of ES (enzyme-
substrate) complex and low catalytic activity.
• Scientists have done intensive work to overcome these problems by developing engineered enzymes by
recombinant technology and rational enzyme design.

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 Medical and clinical applications
• Protein engineering is accomplished to achieve second generation recombinant protein having considerable
properties in medical and clinical applications.
• Mutation, DNA shuffling and recombinant DNA approach were used in protein engineering to get superior results
of therapeutic protein.
• Afterward up-gradation in protein engineering led to fabrication of secreted therapeutic proteins. Eg Interferon,
insulin
• Application of combinatorial proteins for therapeutics and also advancement in gene therapy by inducing
recombination applying meganucleases and DNA double strand breaks.
• Up-gradation of therapeutics for combating against cancer is the major area of interest in protein engineering.

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 Protein engineering in nano-biotechnology
• Nanotechnology was not receiving substantial credit for their difficult synthesis and assembly in functional
systems.
• Then after, a phase came with the studies on biomolecular structural organizations revealing their hierarchical
arrangements from nano to macro levels.
• Proteins, lipids and carbohydrates are the biological macromolecules, being used for biosynthesis of tissues under
synchronized gene expressions.
• Proteins are the most noteworthy amongst them as they are the structural constituents during tissue formation
and aid to the transport and arrangement of building blocks and accessories.

74
 Protein engineering in nano-biotechnology
• Nanotechnology Pioneering proteins recognized as affibody binding proteins, being of non-immunoglobulin (Ig)
origin have been developed employing protein engineering techniques.
• They have high affinity and thus are potentially considered in diagnostics, viral targeting, bio-separation and
tumor imaging as well.
• For development of novel biosensors for analytical diagnosis, insertional protein engineering has been noticed to
immerge during a decade.
• The amino acid succession and organization in a protein affects its conformation as well as function.
Consequently, the capability to transform the sequence and thus the structure and activity, of entity proteins in a
methodical fashion, explore many opportunities, both scientifically and for exploitation in bio-catalysis.

75
 Computing methods have been used to design a protein with a novel fold, named Top7, and sensors for
unnatural molecules.
 The engineering of fusion proteins has yielded rilonacept, a pharmaceutical that has secured Food and Drug
Administration (FDA) approval for treating cryopyrin-associated periodic syndrome.
 IPRO, successfully engineered the switching of cofactor specificity of Candida boidinii xylose reductase.

Increasing the stability and Biological activities -
a) By addition of Disulfide bonds -
1. Lysozyme activity increased by applying disulfide bond at 2,4 and 6 Amino Acids
2. Xylanase - This enzyme uses in paper industry of its activity increase at high temp. by introducing disulfide
bonds ať (1,2, & 3)
b) By reducing Free sulbhohydwyl group- some times free sulphahydryl group decrease the activity of Enzyme
Eg. In human B- intereferon, introduced of serine in place of cysteine reduces free sulphohydayl group

Increasing the stability and Biological activities -
C) Single amino acid acid changes –
Eg-
Alpha1- antitrypsin (This enzyme inhibit the activity of Elastase, that damage lung tissue) – so in alpha1
antitrypsin methionine amino acid (358) is replaced by valine so its activity increased.
Hirudin - Canticogulant - By replacing asparagine ('47 Position with lysine, increase the potency of Hirudin.

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