Protein engineering is the process of developing useful or valuable proteins. Protein Engineering is a second generation of recombinant DNA technology. It involves altering cloned DNA in vitro by novel mutational technique so that translated proteins have slightly altered properties.

1. PROTEIN ENGINEERING
PREPAREDBY
MS.ASHVINI V. SOYAM
ASSISTANTPROFESSOR
DR. RAJENDRAGODE INSTITUTE OF PHARMACY, AMRAVATI

2. PROTEIN ENGINEERING
 Protein engineering is the process of developing useful or valuable
proteins.
 Protein Engineering is a second generation of recombinant DNA
technology.
 It involves altering cloned DNA in vitro by novel mutational technique so
that translated proteins have slightly altered properties.

3.  It is an “modification of Protein structure with recombinant DNA technology or
chemical treatment to get a desirable function for better use in medicine, industry
and agriculture”.
 It is an one of the most exciting aspects of genetic engineering and consist of
designing, developing and producing protein with improved operating
characteristics.
 The techniques like site directed mutagenesis and gene cloning are utilized for
these purpose.

4. OBJECTIVE:
 To create a superior enzyme to catalyze the production of high value
specific chemicals.
 To produce enzymes in large quantities.
 To produce biological compounds (synthetic peptide, storage protein
and synthetic drugs)superior to natural one.

5. PROTEIN ENGINEERING

6. SITE DIRECTED MUTAGENESIS
What is Mutation??
 In molecular biology and genetics, mutation are changes in a genomic sequence.
 Mutations are caused by radiation, viruses, transposes and mutagenic chemicals
as well as errors that occur during meiosis and DNA replication.
 Site Directed Mutagenesis: Also known as Site Specific Mutagenesis or
Oligonucleotide directed Mutagenesis is a molecular biology technique often
used in bio molecular engineering in which a mutation is created at a defined site
in a DNA molecule known as plasmid.

7. SITE DIRECTED MUTAGENISIS
 It is the technique for generating amino acid coding changes in the
DNA(gene). By this approach, specific(i.e. 'site-directed")change (i.e.
"'mutation') can be made in the base (or bases) of the gene to produce a desired
enzyme
 A large amount of experimental procedures have been developed for directed-
mutagenesis of cloned genes.
 A synthetic oligonucleotide complimentary pair to the area of the gene
interest, but has the desired nucleotide change.
 Oligonucleotide- short piece of DNA, usually 10-30 nucleotide long.
 Site directed Mutagenesis is done by using: M3, plasmid DNA, PCR, Random
primer, degenerate primers, Nucleotide analogs, DNA shuffling.

8. SITE DIRECTED MUTAGENISIS
 Occurs in 3 ways:
I. Base pair substitution
II. Insertion of nucleoside
III. Deletion of nucleoside

9. Methods of Site Directed Mutagenesis:
1. Single primer method OR Oligonucleotide Directed
mutagenesis:
 With plasmid
 With M13 phage
2. Cassette Mutagenesis
3. PCR-Amplified Oligonucleotide Directed Mutagenesis

10. Single primer method OR Oligonucleotide Directed mutagenesis
OR Oligonucleotide Directed mutagenesis

11. Single primer method OR Oligonucleotide Directed mutagenesis
 The primer used in this method is a chemically synthesized oligonucleotide
which is normally 7- 20 nucleotide long.
 It is complementary to a position of a gene around the site to be mutated.
 But it contains mismatch of or the base to be mutated.
 The starting material is a single stranded DNA (to be mutated) carried in an
M13 phage vector.
 On mixing this DNA with primer, the oligonucleotide hybridizes with the
complementary sequences, except at the point of mismatched nucleotide.
 Hybridization (beside a single base mismatch) is possible by mixing at low
temperature with excess of primer and in presence of high salt concentration.

12. Cassette Mutagenesis

13. Cassette Mutagenesis
 In this, a synthetic doubles stranded oligonucleotide (a small DNA
fragment i.e. cassette) containing the desired/requisite mutant sequence
is used.
 This mutagenesis is possible if the fragment of the gene to be mutated
lies between two restriction enzymes cleavage site.
 This intervening sequence can be cut and replaced by the synthetic
oligonucleotide (with mutation).
 The plasmid DNA is with restriction enzyme.
 Cassette mutagenesis dose not involve Primer extension by DNA
polymerase.

14. PCR- Amplified Oligonucleotide Directed Mutagenesis

15. PCR- Amplified Oligonucleotide Directed Mutagenesis
 In this technique, first the target DNA is cloned on to a plasmid vector and
distributed into two reaction tubes.
 To each tube, primer is added.
 One primer (A in tube 1 and C in tube 2) is complimentary to a region in one
strand of the cloned gene except for one nucleotide mismatch (i.e. the one
targeted for a change).
 The other primer ( B in tube 1 and D in tube 2) is fully complementary of a
sequence in the other strand with in or adjacent to the cloned gene.
 The placement of primers for hybridization (with DNA strands) in each tube is
done in opposite direction.

16. PCR- Amplified Oligonucleotide Directed Mutagenesis
 The PCR technique is carried out for amplification of the DNA
molecule.
 The products of PCR in two reaction tube are mixed.
 The DNA molecule undergo denaturation and renaturation.
 A Strand from one reaction tube (strand A) hybridizes with its
complementary strand from other reaction tube (strand C).

17. Applications…
 Used to generate mutation that may produce rationally designed protein
that has improved or special properties.
 Investigative tool: specific mutation in DNA allow the function and
properties of a DNA sequence or a protein to be investigated in a rational
approach.
 Commercial application: proteins may be engineered to produce proteins
that are tailored for a specific application
 Examples: in laundry industry- commonly used detergents may contain
subtilize in whose wild-type form has a methionine that can be oxidized
by bleach, inactivating the protein in the process.
 This methionine may be replaced by alanine, thereby making the proteins
active in the presence of bleach.

18. Approaches of Protein Engineering
1. Increasing Stability and Biological activity of Protein:
 Addition of disulphide bond- increase thermostability of enzymes. E.g.
T4, lysozyme, xylanase
 Changing asparagine to other amino acids- thermostable enzyme with
improved biological activity. E.g. triose phosphate isomerase-
asparagine is replace by isoleucine or threonine to have thermostable
enzyme.
 Reduce free sulfahydryl group- to improve stability and activity. E.g.
human interferon.
 Single amino acid changes: improved stability and activity. E.g. alpha
1 trypsin- oxidative resistance enzyme created

19. Approaches of Protein Engineering
2. Improving kinetic properties of enzymes- with the help of site directed
mutagenesis.
E.g. Substilisin, asparginase RE
3. Protein engineering through chemical modification: protein cross linker
glutaraldehydes used in stabilization of protein in solution, E.g. insulin
hemoglobin, lactate dehydrogenase
4. Protein engineering using gene family: isolation of gene from known
family-DNA shuffling- creation of hybrid of different combination.

20. THANK YOU!!!!!!!