Unit-I: Brief Introduction to Protein Engineering
Proteins have important role in physiological processes and they are iinvolved
nvolved in movement,
catalysis, recognition, regulation etc. Moreover, proteins also have several therapeutic and industrial
applications. Advances in Molecular Biology have enabled us to manipulate DNA and express a foreign
gene in other organism (hetrologous
logous expression). This has made advancement in the process of making
changes in proteins at genetic level. Proteins are not always optimized for their properties for various
applications and usefulness of a protein may be limited by low stability and /or undesired substrate
specificity.
Protein Engineering is the process of developing proteins with desired function by
manipulating stability and specificity of a protein. There are two main approaches for protein
engineering, rational design and directed ev
evolution (irrational design).. In case of rational design,
knowledge of the structure and function of the protein is taken into consideration and a rational gene
mutation is planned (Fig.1). Mostly, this is done by making rationally designed changes in the gene
g of the
protein cloned in expression vector of hetrologous expression. The production of protein molecules is
altered by site directed or site-specific
specific mutagenesis of their genes. However, in some cases protein
structure is not available and directed ev
evolution
olution method is required. In this method, random changes
(mutation) are done in the protein and a mutant form with desired properties is chosen.
Figure 1: Two different approaches of protein engineering.
Onee very simple example of rational protein design: Designing an inactive form
fo of Pancreatic
ribonuclease A. Pancreatic ribonuclease A is an enzyme comprising of 124 amino acids that cleaves the
covalent bonds that join ribonucleic acids (RNA). From availabl
availablee literature about the protein we know
that histidine at position 119 is necessary for catalysis. Thus, if at position 119 in the sequence the
naturally occurring histidine is replaced with an alanine, the mutant protein is referred to as a histidine
119 → alanine (H119A) mutant of ribonuclease A. This mutant protein is expected to have little or no
biological activity, because histidine 119 is important for catalytic activity. A simple example of mutation
is shown in Fig. 2.
Figure 2: Diagrammatic representation of a designed mutation in protein at DNA level.
Another example of rational protein design: A very common example of protein engineering is
engineering of enzyme subtilisin,, a protease (a protein
protein-digesting enzyme). To improve the efficiency of
laundry detergents, subtilisin is added in the dete
detergent. However, subtilisin is inactivated by bleach.
Experimental and structural analysis revealed that this inactivation was due to oxidation of the amino acid
methionine at position 22 of the subtilisin molecule. Using site
site-directed
directed mutagenesis technique the
subtilisin gene in E. coli was mutated and methionine was changed by alanine. This engineered subtilisin
showed high activity and stability, and now many laundry detergents contain cloned, engineered
subtilisin.
Methionine is immediately adjacent to a catalytic serine residue and therefore is located at a particularly
sensitive structural position. Methionine oxidation results in increased side chain bulk or perhaps even the
introduction of strongly electronegat
electronegative
ive oxygen atom(s) in the immediate vicinity of the active site
negatively effects the catalytic activity.
PROTEIN ENGINEERING BY DIRECTED EVOLUTION
Let us assume a condition where a researcher has to design a protein which binds best with a given
give
receptor or biomolecule. Let us see how directed evolution can help in this designing.
Site-directed mutagenesis
Steps in site directed mutagenesis
OTHER METHODS OF DIRECTED EVOLUTION: The other methods of directed evolution are
error-prone PCR and DNA shuffling. The gene of protein is mutated using these two methods and
mutant with desired properties is selected and amplified using appropriate methods as we have seen in the
case of phage display technology.
Error Prone PCR: We shall discuss about polymerase chain reaction (PCR) in detailed during module 7
of the course. Error prone PCR is a variant of PCR. Error prone PCR is a technique used to generate
randomized genomic libraries. It allows the initiation of DNA amplification, starting with tiny amounts of
parent molecule, and produces considerable amounts of the mutated gene. The working principle of this
technique is based on the ability of Taq polymerase to anneal incompatible base-pairs to each other,
during amplification, under imperfect PCR conditions. Under these conditions, the polymerase makes
mistakes in the base paring during DNA synthesis which inculcates errors in the newly synthesized
complementary DNA strand. The frequency and number of errors introduced into the sequence can be
regulated by carefully controlling the buffer composition. For proper functioning of this technique, it is
important to use a Taq DNA polymerase which lacks proof-reading ability, because use of a proof-
reading DNA polymerase in an error prone PCR reaction will result in the automatic correction of the
mismatched nucleotides, therefore any mutations that were introduced during the reaction will be lost.
This method has proven useful not only for generation of randomized libraries of nucleotide sequences,
but also for the introduction of mutations during the expression and screening process in the mutagenesis
step.
Webpage: https://www.youtube.com/watch?v=d6UwRUq1bsc
DNA shuffling: DNA shuffling is also known as sexual PCR.. Before beginning with DNA shuffling, a
pool of closely related molecules, with different point mutations, is prepared either through error-prone
error
PCR or other mutation techniques such as oligonucleotide
oligonucleotide-directed mutagenesis.
Various steps involved in DNA shuffling are as follows:
Step 1: Breakage
eakage of molecules into random fragments using DNase I which can randomly create
nicks along each strand of a DNA molecule.
Step2: Sampling of fragments of lengths within a certain range.
Step 3: Further these sampled fragments go through PCR withoutt added primers.
primers There are three
steps in the PCR process without primers.
Step 3A: Denaturing of double
double-stranded
stranded fragments by increasing the temperature, so that
double stranded DNA are separated completely into single
single- stranded ones.
Step 3B: Annealing
ling by lowering the temperature, so that single
single-stranded
stranded fragments anneal to
other fragments overlapping by a certain number of bases that are complementary at the
overlapping region. Once annealing step is over, homologous templates prime each other to
form 5’ and 3’ overhangs.
Step 3C: Polymerase extension by increasing the temperature optimum for DNA polymerase
to extend the 5’ overhangs using the other annealed strand as template. The 3’
3 overhangs are
not changed as DNA polymerase can only extend fr
from a 5’ end.
The three steps of denaturation, annealing and polymerase extension are repeated for multiple cycles.
Diagrammatic representation of DNA shuffling is shown in Figure 5.
Figure 5: Diagrammatic representation of DNA shuffling (Please go through the vedio:
https://www.youtube.com/watch?v=hWHqFIi4jM0)
In each PCR cycle repeat the average fragment length increases. After many cycles of PCR without
adding primers, molecules of original size are expected. Recombination occurs when a template from one
molecule primes a fragment from another molecule.
The purpose of DNA shuffling is to recombine beneficial mutations from different molecules, and obtain
molecules with even increased function. Further DNA shuffling improves the search of local fold space
by means of a random yet correlated combination of homologous coding fragments that contain limited
numbers of beneficial amino acid substitutions.
Note: You can visit
https://www.slideshare.net/bansalaman80/protein-engineering-40209038
