discriminate

sOurcehuman analyzed (i.e., according

ribotyping) should when
firstthe 254

The Ribotyping pig, The

ofvs. The have
coniamination PaleoWater
eo services
throughhorse, to
given
animal Paldifferent
its the
Water indistinguishable
own enzyme
gives
multiplehumans, hasexperience enzymes
strains
DNA
spccics-specific
for specific
Division also
of E.
fingerprinting of coli DNA etc.). been provide
E.coli. of
E.coli.contamination "gcnctic the
analytical
of Afler used
PalcoScience GENE the
DNA sources to
fingerprint". identify a best
service techniques.period consortium.
fingerprinting from information (for
of particular
(E.coli
contamination. offers ofpatterns,and
humans incubation,
IDM) on Each En
using and a strain strains ymes
routine more
when typing
"Ribotyping" has animal the
of of used
excellent E. E.
sources. basis coli coli coli E.purposes: as
discrimination
for
strains
needed.second
is
precision DNA specific
can This zach best
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help service particular discrimination,
to for extracted animal
identifying can
know can
animal grouns be
the also (ie. and used

MODULE 36

utations
knowledge_ in make to
copying proteins.
existing
nature.modifyingscientists protein It 36.1 3¬.6 36.3 36.2 36.1
36.5 36.4 Introduction.
proteins
natural is structural translated involves
1954, each
Protein Introduction ChimericF.otein
Protcin Examples
36.5.736.5.6 36.5.5 36.5.4 36.5.336.5.2 36.5.1 Objectives
TechniquesProtein
Protein 'rotein
Max foods,other. Recentchemistry,
on when tostability proteins
altering
protein ofPerutz engineering advances engineering engineering
engineer Purification
Protein Application
relationship. Change
Improvement Engineering
Improvement
drugs The enginecring Proteins of
protein of of
PROTEIN
today'sbegan of havecloned Protein ENGINEERING
Protein
and
successes diffiçult, enzymology engineering
engineering Protein
final protein, of
structure proteins. in in of catalytic
advancedchemicals. the and is is of
structure. absence enzyme protein Engineering
in in Strategies.
Engineering.
can thereforemolecular mergingslightly DNA a
second Engineering.
stability
catalytic
work in
genetic enzyme.
was protein be Design, and altered in applicationsfor
applications activity,
in of made
engineering
algenetic
sodirection engineering regarded protein biologystructural ofgeneration vitro efficiency. of
production several Gene enzymes.
enginecering properties. easier substrate Applications.
limited. by
techniques as
engineering and disciplines novel Homology. to
of hormones. by to
ofbiopharmaceuticals.
redesigning related chemistry specificity
But protein are and recomtbinant understand
protein Biauhoology
Gere
wiT mutational
hewere the analysis
studied
engineeringtransformfields techniques
mainly like engineering
to of
not nature molecular
catalytic
alteror technique DNA enzymes.
structure
the complementary of
available concerns
as the rather altered, now technology.
effects way biology,
early so activity
than allow
and as we with non that
of
 these
Jome ofþrokin
I3olntdd
prvin? Aaiend Engihetring
ProkinPrelein
g anging,th Pron
/Prokins |}
th
muagenesl
Random Prsrin foon em nveluinyin
(frein iJby. th
the auih orgahism. gin eing
design eeing atin4
rsyme Novel En
| s
inbat PoeperhiesJ qin
eering and isUoned a
order s
mergng Stlono
used
table INahene for vA
b aleod
gemekon
of
mate and to
heerenl ioyiho
it aahe Lachie at dynthesis fropiahto
indepeo Obje by o
in nbohak
bindingIhe chiveo dseplind borelrecom Colm
dunt bon he of
degradahon -0
nal«onh binn
tgho ensme mitasonN
of Inioeosing lke
DNA
tofydhor fo Herma/olwranA moe
olrnt. Ake mn
cn
Jubob genesca, behe
for ik
maby
l
rtfomt rate
esithank
256
Gene Biotechnology
Protcin engineering in today's state of development can be defined as modifications of prote
either by mutagenesis or by recombinant DNA technology or by gene modification or by chemical
rcaction.

Today, we study the structure of protein first, think of structure function correlation and then
attempt for favourable modification.
Basic assumptions while atteming protein engincering are:
(a) Proteins have limited number of basic structures.
(b) Minor changes are superimposed on them to give variation.
(c) Only few amino acids are important from function point of view.
(d) Similar patterns of chain folding and domain structure can arise from different amino acid
sequences.
Thus, it is not maný changes in amino acids but only minor changes at specific
may be desirable. positions which

36.2 Objectives of Protein Engincering

Enzymes, antibodies, hormones, receptor proteins, etc. are the candidate classes of
engineering. Out of these, enzymes are of most interest. proteins for
Overall aim of protein engineering application is to get functionaily more
proteins from above classes and of courseeconomic gains from useful, more efficient
will be made clear when we look into them wherever possible. The point
sub-objectives or fields of applications.
Protein engineering can be used to:
(i) increase stability of enzymes;
(ii) increase catalytic efficiency of enzymes;
(ii) introduce new active sites and thereby new catalytic activity in
(iv) alter pH dependence of enzymes; enzyme;
(v) improve thermostability, modify substrate specificity of
(vì) enzymes;
understand structure function relationship for an enzyme in better way -
of actions; useful for predictions
(vi) transfer the ability to make desirable
enzyme in (easy to cultivate, safer to use)
(viii) produce tailor-made enzymes in large organisms;
structural genes quantities by increasing the cxpression of relevant
(ix) produce hybrid
enzymes;
(x) make isolation and
purification of enzymes simpler;
(xi) make hormones resistant to
attack by antibodies or stomach
(xii) get more specific, more potent enzymes;
(xiii) get humanized antibodies biopharmaceuticals altered pharmacological action;
with
(chimeric) with less immunogenicity.
 257
Protein Engincering

36.3 Protein Engineering Strategies

There arc two general strategics for protein engincering. The first is known as rational design,
in which the scientist uses detailed knowledge of the structure and function of the protcin to make
site-directed
desired changes. This has the advantage of being generally inexpensive and easy, since that detailed
mutagenesis techniques, are wvel-developed. However, therc is a major drawback in
structuratHknowledge of a protein is often unavailable, and even when it is available, it can be
extremely difficult to predict the cffects of various mutations.
Computational protein design algorithms seek to identify amino acid sequences that have low
energies for target structures.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. Using for
computational methods, a protein with a novel fold has been designed, as well as sensors
un-natural molecules.
The second strategy is known as directed evolution. This is where randon1 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 applicd. This method mimics natural evolution and
generally produces superior results to rational design. An additional technique known as DNA shutiling
mixes and matches pieces of successful variants in order to produze better results. This process
mimics recombination that occurs naturaliy during sexual reproduction. The great advantage of
directed evolution techniques is that they require no prior structural knowledge of a protein, nor it
is necessary to be able to predict what efiect a given mutation will have. Indeed, the results of
directed evolutiYn experiments are often surprising in that desired changes are often caused by
mutations that no one would have expected. 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
screened for.
equipment to automate the process. Furthermore, not all desired activities can be easily
Rational design and directed evolution techniques are not mutually exclusive; good researchers
will often apply both. In the future, more detailed knowledge of protein structure and function: as
well as advancements in high-throughout technology, will greatly expand-that capabilitis of protin
that
engineering. Eventually even unnatural amino acids may be incorporated thanks to a new method
allows the incorporation of novel amino acids in the genetic code.
36.4 Techniques of Protein Ergincering
Techniques used for protein engineering fall in two basic categories: (1) genetic modifications
and (2) chemical modificationg.
3-dimensional strucure of an enzyme is dependent on amino acid sequence (i.e.. primary
structure) and the environment (i.e., pl4, ionic strength, temperature, ete.) in which enzyme is placed.
3-dimensional
Therefore, changes in amino acid sequence (i.e., primary structure) will be reflected in
structure of enzyme and its properties. This is the basis of main strategy used by protein engineering.
Chemical Modifications
method is most
() Functional group on side chain of natural enzyme may be changed. Thismutagenesis.
widely used and had more importance before advances in site directed
 the " - - Techniquu
Moed
ehajTheminervaclegae Mutant smipldaDNA Lor Mukehi Mutabons
thonges Site 0 n
Plamid Hinda on Onodifiahsn
Coseheqeneh direelombinah on ehemitedGenehr
ekpNA direted
Tube O leavage cted is Can in
JTho. Tanyom LcoRI mtag en modpahony
colj E mutagensrs o
moftrsl1 definedihvolvemobptafony
Meovage
Mutogehesis
genbmit an
Jequenie /
tuns prorio
eseith
eslarglplutement :ikþrorin
L a segu :-
mutaht anm}thaage Jechons en :-0
can oryanm In
NormPim lnyineertng
diteted Pal!
ohde DN be molee thange
be o
cut depin in. of-oyinal ofelar
Ot he oNg DN.magenteis
tod Hhe Hindnan
ueleohdesta
oliyonl ed o
nuelerc bioloyy
gene mattridbeomin
an
op hin
eplacecl to muk odeeminee
locakan.
peape ad
prori
ant ÉCORI inlado
btait
be
utted
'mby D
NA frop )
he
in
Tube 2 heTesired lotall'zel(akag
or',
baaic
sed orgeéic
aud
7ohekr Ue olyonuleohle biranidousleed
fragmentSyn4hehr
esbh
tuso
ieo
Tanboo
oliyonbeteen mwtent

uulaoble la
snall
oNA
258
Gene Biotechnology
(2) Total chemical synthesis of an enzyme is only a theoretical possibility. Practically, it is
expensive and knowledge also is still insufficient. Semi-synthetic route is adopted today
Here. protein enzyme is cleaved to shorter reptides. These shorter peptides are then modified
or replaced and reconstruction of protein enzyme is done. For example, chemically changing
serine residue involved in catalytic activity of enzyme subtilising into cysteine residue
resulted in major change in catalytic activity.
Chemical modification approach has its advantages and disadyantages.
Techniques

Genetic Modifications Chemical Modifications

Site directed Localized random Furctiona! Parts of original protein

mutagenesis mutagenesis group on niodified and replaced
side chain
) f 3-D struc:re is f3-0 structure is is changed
known and structure ot known and molecular
activity relation is sequence also not known
knovn.
or or

("'}I3-D structure is (l1) If 3-D structure is

not known, but homologous knovn. but structure-activity
sequences are known. relation is not known

(Discussed in Moduie 27)

Advantages
(1) Attachment of co-cnzyme to enzy me is possible.
(2) Thioprotcase activity of papain changed to oxidoreductase.
(3) It can be applied in conjunction with genetic methods.

Disadvantages
(1) It is applicable to amino cids that have reactive side chains and only certain alterations
can be made.
(2) Only amino acids at surface of protein are available for modifications.
(3) There is little specificity. Modification of single amino acid is difficult, e.g., if histidine is
heing modified, all histidines on surface will become target.
Chemical modification of surface amino-groups (13 out of 15 available) of enzyme a
chymotrypsit1 resulted in upto 120 times greater stability of enzyme. (Alkylation or acylation was
done using acrolein or succinic anhydride respectively.) Also lactic dehydrogenase could be made
resistant to attack by trypsin by treating it with methyl acetimidate.
36.5 Examples of Protcin Engineering Applications
36.5.1 Improvement in Stability of Enzyme
Thermostability of an enzyme could be incrcased by following substitutions:
 259
"Protcin Engincering

Lysine substituted by Glutamine or arginine

Valine sut tituied by Threonine
Serinc substituted by Arginine
Aspartate substituted by Glutamate

Isoleucine substituted by Threonine

Asparginc substitutcd by Aspartate

Glycine or sering substitutcd by Alanine

Glucose isomerase enzyme is used to convert glucOse into fructosehile making high fructose
corn syrup (HFCS) for spft drink industry. This enzyme c¡n be, made more efficient by improving its
propertics. Replacing lysing with argiline or glycine with alaninc gives increased thermal stability.
In glucose isomerase, lysine at 253 position is replaced. Hydrogen bonding increasçs.
Stronger s-s bonds also incrcase stability of enzyme. Isoleucine in 3rd position of T, lysozyme
Half-life
is replaccd by cysteine. Cysteine at 3rd position and at 97th position then form s-s bond.
of enzyme at 67°C increases from 1I minutes to 6 hours.
Thermostability of a protein cn be increased by creating a molecule that will not readily unfold
at elevated temperatures. Such thermostable enzymes are also often resistart to denaturation by
organic solvents,extremes of pll andproteolysis. Aspargine, glutamine with high temperature undergo
deemidation resulting in localized changes in folding of peptide chain and loss of activity. Converting
aspargine to threonine or isoleucine therefere results in enhanced thermostability.
Protease enzymes that have been genetically modified for greater stability are alrendy in market.
Proteases now used in detergents are oxidation resistant proteases. (Oxidation may be causcd by
bleaches (e.g., H,0,) inactivation of enzyme is the result). Oxidation resistant proteases were first
developed by Genentech in 1982 by site direted mutagenesis. Subili_in froin Bacillus amyloliquifaciens
with methionine at active site at 222 position replaced by alanine was obtaincd but it had only 80%
of the speccific activity. Later, serine or glutamine in place of methionine gave 100% of the specific
activity of wild type. Storage stability of these enzymes is greater than that of the original wild type
enzyme.

Protein Engineering 'of Laundry Detergent

The enzyme subtilisin is aprotease (a protein-digesting proein) produced by bacteria, and has
efficiency of laundry
a broad specificity for proteins that commonly soil clothing. To improve istheinactiviated
detergents, detergent manufacturers added subtilisin. However, subtilisin by bleach,
and it was found that this inactivation was due to oxidation of the amino ac1d methionine at position
22 of the subtilisin molecule. Using site-directed mutagenesis of the sub:ilisin gene in E.coli, this
methionine was charnged to a variety of other amiio acids, and the subtitisin activity and bleach
resistance of the mu:ated protein was tested. It was found that substitution of methicnine by alanine
Was the best in terms of activity and stability, and now many Jaundry detergents contain cloned,
genetically engineered _ubtilisin.
Protein Engincering of Subtilisin
Generation of a subtilisin with a half-life at 65°C that is 50 times greater than wild type of
recombining segments from five diferent subtilisin variants. Method used for achieving protein
engineering is staggered extension process (StEP) [Fig. 36.1]. This also relies on repeated cycles of
 nuturn
enajme -A - -
Dsedvantages:-
ahons DassibilihchemitalOhr The The
mutugchei
busdPR
dhn oNA Changd
PeR
an
be Práhalh melde
modifabonuthion Fr
0 ube ulesmhnigue
rHhe onlymade It ae Mut kaps -fhud A
amin s lndergo
1sLstrahl ant
Thany ShrandA
peajriy ppliable be one is
aid nd (penatn
experchanq
auda s heahon
Plaam o
ehoils c)
ef
she denakwrahon idi E
ikens out
toli
aat ca- d.
ensyrhe and hub
Jupale min 0Tohad for
reochen
e
lSrandn]
cauda Kouledehemital atnplifakon -Shand D
to and chahgid Shnd
of that ehaymethenial hukes direcien
f he -
dingleprohin hove mad'abons so Sand
The Complen
hjhdl
tyanl dre
is
Synis oN A onuleohde
pla muag SiHe Sre
is
is of Jran
jb
uninud erreachve eer possihlz dhi renalugah'on $ee mired. he umnt ontory u'h dehed
utcd dirtddirehd
s enesrs
urail
able înautiwint. o! D b eA in
drde Funwona/ an isth in' of or of on
mole pms te ko mn mutag
is enaym tah yomesi_cdodja 9
hulo
elhnngue
dt; chain aht o
for lomplemen
emplementay hibe eguene plastmd bjons
e DNA rearh'on eneois
Lus gooups for k
modrhakonsandont 13 he t :-
hybrdiakien
mnole cdone - mim The i-
ontary don in vetor
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 Gene Biotecchnology
260

However, in the StEP process, one starts

Imelting, annealing, and extension to build the variant genes.
with a mixture of full-length genes, denatures them, and then primes the ynthesis of complementary
strands. After ashort period of primer extension, the will DNA is subjected to a round of mclting.
anncal to templates with a different base
annealing and extension. Some of the extended primers more cycles of extension, melting and
sequence and on further extension willgenerate chimeras. The
produced.
annealing the greater the variability that can be

Denature and
anneal primer

DNAextension
for short period

Denature and
anneal

or

DNAextension
for short period
h

Multiple cycles of
DNAextension for short
period, denaturation
and annealing

1!

Fig. 36.1:The StEP method for generating hybrid proteins.

Ahybrid gene will be constructed from two homologous genes.
36.5.2 Improvement in Catalytic Efficiency
stearothermophilus had been
Three-dinmensional structure of tyresyl t-RNA synthetase from B.
already studied and active site is mapped. With computer graphics, predictions can be made about
site. Gene- for enzyme can be modified by
effects of changing one or few amino acids of active changed and catalytic
oligonucleotide directèd mutagenesis. Affinity of an enzyme for substrate can be
efffciency can be imoroved.
Protein Engincering 261

Single
strands
from
horn'ogc.
genes Synthesize
complemnentary
strand with dUTP
instead of dTTP

DNase

Uracil

Mx and anneal

Exonucease
+ DNA palymerase
" DNA igase

Endonuclease V

Convert to doubla strands

Fig. 35.2:The RACHITT method for creating hybrid proteins

 anh'nyain soaleInkrran
fre 3Fabiliy, bends Ty
Add;hon
Thkeduung ahhhYpsin
godmethibnine )
i Hum anqlutamine Chunying om
Inreehy
-Thi ple:Eym4 Appliakons DNA -
lge uls Reduunachyft.
bioloqi ta mukelysojmi-
shubiy gwthydry U metaionine merisl
tpa ahino bo ' ehinin m Sequehce
thurmo
dhubye i make of
mogen intore and disuF7e
,has aal th eundeyo
sparaqine
Hhe aminU
4ingle ahd
binds b its it by
b tod nhihits the he t Yy ma y
ehalppiatonA or
be ehanie:qroups aLhihy hanging o
makes Thi
oubychyl
:/is es Jutle bons becu prouin
y biolagita elastase prduud deamidahion
i3 can to veploed bewd
i-
ahaor1mporkl a dam4ges
elathase
improre and due oher ah
:Inveasu
2-4.6 odi'zed laundry
ud achon
be
Preins o
aLhre by ba
h dubobhhon, ahviy.duboh hhIPS nd mate b gronps
increased amino Joodamino alanin in
:- neuttophi)
pretnbs of Form dergents veahzak
be
rred
dmin and atbiolo moyke
Wsd Rhons aud:
humAhoin - hrgh qral auds Humhercby by
o s 4n
Slfhydyl fom femperahr J}abilih bleoch
lyse insulins oor of by A b nginerel in
ahon achvity
ysttine many my a
ínhihihor elaaintepron
preduung
yoejn fem hryhjh rahon
blod in
Femperaure : of achContain
ere enperaure feme to
In .e roups enaymts.
vrdues t
. vahng preduce ad
lots thio heplaing he Inhoduson
foud n f h SubHl1sepraduce
dhseaoe eloise moh morl hunber aa/ T
tDue dra
process dnd uces aminoot he
to ohabe and proeins
fo be ysteine
aminoy.
enist of and p ro protinu
In
i dauas it pron of octive /n
Jughydryi gluamie forming uhose
gek as drauide ln halhal
il's lth lld
cleaed cdimyochvihy.
pr
gine in he Ore
Juna doujide ),
l. he pro
rtr serine qroups
ad bonds 2,3
cess. hpetuilored
es tnd preshe
hexamty Jerine
dsujte
Hdues olrgo prin
he-loss and a formhas
hal- L,t h
an mrs og
 Gene Biotechnolopy

262 wih
restriction endonuclease enzymes) is possible
(c.g., combined with
Production of site-specific nucleases
recognition and binding properties of proteins is'5-iodoacetamide.
Here, DNA modified using
protein engineering. 128 of E.coli CAP protein, has been
clcaved DNA at the centre.
chemical cleavage agents. Cys DNAcleaving agent that recognized and recognizing upto 20 bases
yiclding a restriction enzymes
L10-phenanthroline' give
the recognition site (22 bp) for CAP. This may
for isolating long DNA fragments needed for
of useful
instcad of 6 or 8 bases, and may therefore, be
scquencing and mapping.
a,-antitrypsin (AAT) devclopment of emphysema
Protein Engincering of responsible for the
thought to be against elastase
Cumulative damage to lung tissuebyis loss of lung elasticity. The primary defense
characterized acids.
an irreversible disease glycosylated serum protein of 394 amino
(AAT), a brcakdown
damage is a,-antitrypsin its genetic deficicncy leads to a prematurc
neutrophil
known because betwcen AAT and
The function of AAT is individuals, there is an association 359.
of conncctive tissuc. In
healthy
methionine residue 358 and serine residue
cleavage of AAT between concentration
elastase followed by smoking results in an increased
emphysema, because elastase. In addition,
Smokers are more prone toconsequently increased exposure to neutophil ..ethionine sulfoxide.
and 358 to
of leucocytes in the lung free radicals and these can oxidize methionine fit into the active site of
leucocytes liberate oxygen than methionine, it does not
is much bulkier mutagenesis, an oxidatior
Since methionine sulfoxide means ofsite-directed
a poor inhibitor. By valine.
elastase. Hence, oxidized AAT is constructed by replacing methionine 358 with
been
resistant mutant of AAT has AAT was an effective inhibitor of elastase
and
inflation, the modified
In a laboratory model of
oxidation.
was not inactivated by
Dibenzothiophene Mono-oxygenase modification of
Protein Engineering of of diesel can be achieved by
biodesulfurization
Increased rate and extent of here is RACH ITT (randor
Method used for protein engineering
dibenzothiophene mono-oxygenase. 36.2) (a method conceptually similar to the original DNA
(Fig.
Chimeragenesis on transient templates)produce crossovers).
chimeras with a much larger number of DNA
Shuffling method but is designed to generated from onestrand of all but one of the parental
fragments are on the annealing
s method, the gene reassembled on the full length to a greater or lesser extent molecules will b
These fragments then are parental
there is always a chance that the
DNA sequences. Thus, great as expected
of mismatclhed variation generated will uot be as
degree cf
Tecreated preferentially or that the
Specificity of Enzymes
36.5.3Change of Catalytic Activity, Substrate Lawrence had been able to change
th
approach, Kasier and
Using chemical modification new enzyme ca
oxidoreductase activity. Although thisapplication
enzyme papain to of t
uoiprotease activity of the biologically important molecules, commercial
Calalyze the oxidation of certain
enzyme is yet not developed. conversion of mala
altering substrate specificity of an enzyme could be the
best example of Is n
dehydrogenase (LDH). However, this enzyme also
denydrogenase (MDH) enzyme to lactate
commercially useful.
Protein Engincering 263

6.54 Application ef Protein Engineering toUnderstand Structure-Activity Relationship

Although commercial gains from protein engineering work have yet to be reaped, o.e of the
oreatest contribution of protcin cngineering cxperiments has been to get better insight of structure
ivity relationship of enzymes and some other proteins. For such protein, engineering studies both
ile-directed mutagenesis or chemical modification experiments have been uscd. Enzymes like
"hacteriophage 1, lysozyme, Tyrosyl-+-RNA synthetase, dihydrofolate reductase, staphylococcal nuclease,
Iypsin, etc. have acted as model systems for such studies. Role of amino acids, their significance at
snecific positions, non-essential sites, essential sites, stability factors, etc. are understood from such
work.
To consider few examples
(a) Insulin A
and Bchain are linked by C peptide of 35 amino acids but a sequence of 6 amino
acids for Cpeptide is found to be adequate for Achain Bchain linking function.
(b) In cytochrome C, a phenylalanine residue has been identified non-essential for electron
Iranster but is involved in determining the reduction potential of the protein.
(c) An case of tyrosyi t-RNA Synthetase, it has been observed that site-directed mutagenesis at
positions 35, 45, 48, 51 affect overall catalytic efficiency of enzyme and can be improved.
(d) In case of enzyme dihydrofolate reductase (which catalyzes NADPH dependent conversion
of Dihydrofolate to tetrahydrofolate), site-directed mutagenesis and protein engineering
work has helped to understand role of different amino acids in enzyme-inhibitor interaction.
(Inhibitors of enzyme create tetrahydrofolate deficiency, then disruption of DNA synthesis
and ultimately cell death). Inhibitors of DHF and their interaction is, thus, important. for
chemotherapy (treatment of bacterial, protozoal infectionsand leukemias).
(e) From studies on staphylococcal nuclease enzyme (polypeptide with 14 amino `acidss and
without disulfides) in which mutants were obtained to change amino acids at different
positions, it became clear that contribution by aiy particular amino acid may not be
constant but can depend on the presence of other ariino acid substitütions at sites remote
from the original mutation.
36.5.5 Purification of Enzyme Made Easier by Protein Engineering
The presence of many arginine residues in enzyme protein would raise its pl (isoelectric point).
it atC (or N)
If pl for native enzyme is 7, modified enzyme with arginine residues introduced into
terminal will have pl closer to 10.5. Then below, plH 10.5, modified enzyme will carry net positive
and
charge and will be retained by cation exchange resins while above pH 10.5 it carries no charge pll and
would be eluted from cation exchange resin. Thus, cell extract can now be taken at nigher
enzyme would be
most of the proteins would pass through ion exchange column whereas modified
retained. Subsequently, modified enzyme can be eluted by raising pH above 10.5,
36.5.6 Protein Engineering Applications to Hormones
that differed 66% of its
^cientists, have prepared artificial versions of calcitonin hÍrmon
body's defences, speciticaly
components, but still worked. Natural calcitonin can be attacked by the susceptible to attack by the
by the antibodies and is rendered useless. Natural calcitonin is also both stomach enzymes and
stomach's protein cutting enzymes. Synthetic calcitonin is resistant to
antibodies. It is simple to prepare and less expensive.
 Gene Biotechnology
264

and his colleagues at Rockfeller University have already transformed a brain hormohe
Kaiser extensively modified but active site wa
Reta-endorphin which is body's natural opium. Protein was
effective.
left intact and
an example of production of hormone made easier by protein engineering. There are
There is technology. In his approach, gene for Achain of
eforts on to produce insulin by recombinant DNA
and a gene for B chain of insulin or gene for proinsulin are joined with another gene (say
insulin
(B-gal structural gene). Recombinant products are formed as follows:
tryp E or Bgal B chain
Bgal Achain
tryp E,B chain
tryp E, A chain

tryp E Proinsulin

chain or B chain from recombinant product is critical. By genctic engineering

Separation of A E and insulin gene portion. This produced
gal or tryp
AUGcodon is introduced between gene for B A chain.
Methionine
methionine between two proteins, e.g.. gai site and
cyanogen bromide treatment can cause cleavage of protein at methionine example.
Further, In this
Later, A chain and B chain can be linked.
Achain or B chain can be separated. production.
genetic modification helps for recombinant protein
Methionine- A chain tryp E- Methionine -B chain
tryp E
cyanogen bromide tryp E cyanogen bromide
tryp E

A chain Bchain

Insulin

Biopharmaceuticals
36.5.7 Protein Engineering Applications for
protein engineering is aimed at biopharmaceuticals to alter pharmacological action. Making
Much and coupling them to targeting mechanism s
proteinic drugs which are more specific, more potent survival time in patients, reducing th
that effect will be on few cells or cell types, improving their
side-effects are some of the objectives of protein engineering in this field.
therapy, insulin is mostly assembled as zin
Fer cxample, in the neutral solutions used forabsorption. By making single amino acid chang
Containing hexamers. This self-association may limit
are monomeric at pharmaceutic
Brange et al. (1988) were able to generate iIsulins which biological activity w
concentrations. This improved the absorption by two to three times and
preserved.

Similarly, hybrid interferons,combining the aminoterminal half of IFN - a, with the carbox
anti-viral properties from the tw
terminal half of IFN - a, have been prepared which show distinct
parental interferons.
The commercial success will depend on many factors.
 265
Protein Engincering

Endonuclease
cleavage site

Endonuclease
cleavage

Endonuclease IlI
digestion

Digestion with
mung bean
nuclease

Ligation

two related proteins.

Fig. 36.3 : The ITCHY method for creating hybrids of
In the figure, the two related proteins are encoded1 and by genes 1 and 2.
the 3 end of gene 2.
The end result is a hybrid gene comprising the 5 end of gene
 O Celluasc Catalase ing
Hirudin
9 ) Table Ioehve
l4 Sike bydro
by
Fiun
9915) 1) Dijdrofo
Inrease
kataseAuase Gl
lase
ueo
ny Amino0cylascBrom
elain ly3o*]ne folate asp
Proase PeutPapain
in lipese : diretd fol
Rehn 9 aeprolein
Jerehed
rqin by A
Some analog lale
et ase : i- duseoni Thrs
: :
Jurose Indusnl Rplo mutaggedutose: lula
heese i: : meatcheese inrrioh
Whey ue res
ing Uaryng Hh
eherqent,
cheeseDehrq 4ahenidant 6eer AnhoxtWank
Meat
4lcebol meitmaking
alehol
Beer te suh ulh s ysihe
tndeniter, Preparah eness
makiubinhgqabon ment
tenderiser tnd as in 1
frt maing And ehsjmeo of , cataly: Hhe
in on replace meho
Coy
nhes/3
odujon
alatsel -prparasonJocese pryercd îh
qlu erisr glyne pold leecf
jwis jue ,prepared
cosé of
aleoh f lmino hexate
fuie and salivary
bymnt andio gland
prdehor
el le of heof
Wlahel dai hydrahsa foods. duehonpro any an
ple f
f anhahon
þrodhion foeds
Laminequ
dashrabon helr oher of
conrersion
hiudin
ukon
produeton Mavoviny lyinenshiA
bpletops
aminooud auds
by Can
s danlne heand ofbe
gro 7.
in
aminoouts.
ineaed
hlHh &
meaas dihydro
of hrembin
proleio
foundtemor folabe by
Srutaremployi'ncells
Jevd
goGycel The teroinhibitor
prod ls ta S.
hihih'p n
DHFR ue 6,78- folt
desereaes ty
eho rplo
of
n
 Genc Biotechnology

266
Absence of Gene Homology
Chimeric Proteins
in
an absolute requirement for significant
described above have
36.6
gene-shuffling methods there may be a wish to create hybrids betweenthig
The between the parental sequences. However, homology is less than 50%. Achieving
homology functional similarities but whose sequence and the first oneto be developed(Ostermeir
proteins with combining non-homologous sequences
creation of hybrid ei zymes). This method
methods for
requires ITCHY (incremental truncation for thegenerated by the truncation of two template
procedure
al., 1999) direct ligation of libraries of fragments ends (Fig. 36.3). This ligation fragmente
was
e

isbased
onthe
template being truncated from oppositebut the downside is that the DNA
sequences, each point of crossover gene.
homology at the their position in the template
removes any need for to
a way that is not at all analogous using timed exonuclease
reconnectcd in performed
may be
process, the incrementaltruncation was improved process was developed
In the original
ITCHY
digestions are difficult to control. An linkages incorporated at random
digestions. In practice, these are generated with phosphorothioate exonuclease digestion then generates
templates 200la). Complete resistant phosphorothioate linkage.
where the initial gene (Lutz et. al., nuclease
along the length of thedetermine d by the position of the
fragments with lengths and is much simpler to perform.
thio-ITCHY gerne. However,
This method is known
as
they coitain only one crossover perSCRATCHY, it is
ITCHY libraries is that process known as
One drawback of with DNA-shuffling methods, a problem with methods
such
ITCHY libra.ies
al., 200lb). A major insertions
by combining additional variation (Lutz et.non-furiction sequences due to mutations,
possible to generate generate large numbers of three-dimensional structure of proteins, it is clear
as ITCHY is that they
Furthermore, when one examines the
and deletions. domains and motifs. recombine thess
are organized into proteins might be to
that they chimeric developed (O'Maill
attractive way of generating this have been
Iherefore, amore
nove! ways. Two general methods of doing (structure based combinatorial protein
Comains and motifs in Arnold, 2003) and these are SCOPE chimeragenesis).
& (sequence-independent site-directed
e. dl,, 2002, Hiraga
Cgineering) and SISDC