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Pharmaceutical and industrial protein engineering: Where we are?
Article in Pakistan journal of pharmaceutical sciences · January 2013

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REVIEW
Pharmaceutical and industrial protein engineering: Where we are?
Amro Abd-Al-Fattah Amara

Protein Research Department, Genetic Engineering and Biotechnology Research Institute,
Mubarak City for Scientific Research and Technology Applications/Microbiology Division,
Pharmaceutics Department, College of Pharmacy at King Saud University (KSU) Riyadh, Kingdom of Saudi Arabia
Abstract: The huge amount of information, the big number of scientists and their efforts, labs, man/hrs, fund, companies
all and others factors build the success of the amazing new branch of genetic engineering the 'Protein Engineering' (PE).
It concerns with the modification of protein structure/function(s) or building protein from scratch. The engineered
proteins usually have new criteria(s). Engineering proteins can be mediated on the level of genes or proteins. PE fined its
way in different important sectors including industrial, pharmaceutical and medicinal ones. Aspects about PE and its
applications will be discussed with this review. The concept, tools, and the industrial applications of the protein,
engineered proteins and PE will be under focus. In order to get up to date Knowledge about the applications of PE in
basic protein and molecular biology, several examples are discussed. PE can play a significant role in different industrial
and pharmaceutical sectors if used wisely and selectively.
Keywords: Protein engineering; applications; rational design; directed evolution.
Protein they were both awarded Nobel Prize in Chemistry in

Proteins are macromolecules, which participate in every 1962.
process of different cells. Protein name derived from the
Greek word prðtos, meaning “first” or “foremost”. The Rapid advances in the related fields (including DNA
Swedish chemist Jöns Jakob Berzelius was the first to isolation and purifications, cloning, sub-cloning,
describe protein as a term in 1838. James B. Sumner sequencing, site-directed mutagenesis, gene synthesis,
(1926) showed that urease is a protein. Changing protein new expression systems in prokaryotic and eukaryotic
composition is a natural phenomenon, e.g., Sickle cell cells) have provided the molecular biologist additional
anaemia and Cystic fibrosis (Harris, 1992; Platt and tools for modifying existing proteins to improve their
Falcone, 1988). Post-translation modification, which can catalytic activities, stability, and selectivity. Different
happen either before the protein is used in the cell, or as a results and information about protein structure/function(s)
part of control mechanisms is a natural mechanism for have been used by different researcher to improve their
modifying protein structure (Caraglia1 et al., 2004). design. Protein structure/function(s) knowledge has been
found its applications in different fields including food
Proteins work together to achieve a particular function, industry, enzymes, pharmaceutical products, protein
and they often associate to form stable complexes folding applications, targeted drugs, nanomedicine,
(Anthea, 1993). Insulin was the first protein to be hormones (insulin), growth factors, protein crystallization,
sequenced, by Frederick Sanger, who won the Nobel antibody-antigen docking etc. (Goodenough 1995;
Prize for this achievement in 1958. He has succeeded in Runbingh 1997; Arulmuthu et al., 2009; Crisman and
solving the insulin sequence using protein amino acids Randolph 2009; Debbage, 2009; Huang et al., 2009;
sequencing rather than using the insulin related genes. It Sivasubramanian et al., 2009; Kang and Jang 2009).
was a complicated process especially in the presence of
disulfide bonds between A and B fragments. However, Protein is highly specific and could produce complicated
Sanger and Thampson (1953a,b) have identified the processes like DNA and RNA polymerase, where the
importance of the insulin 3D structure. The first three- DNA polymerase reaction is catalyzed by two-metal ion
dimensional (3D) structures for hemoglobin and mechanism in the 3’-exonuclease reaction. In contrast
myoglobin have been resolved by Muirhead and Perutz RNA polymerase do more complicated function while it
(1963) and Kendrew et al., (1958 a,b) respectively. The work in one ribonucleic acid strand only. They are able to
3D structures of both proteins were determined by x-ray initiate RNA synthesis without requiring a primer
diffraction analysis. On the basis of their pioneering work, oligonucleotide exhibit an abortive initiation phase and
*Corresponding author: e-mail: amroamara@web.de

"Any change in the gene/protein lead to a new structure/function(s) is in the frame of PE" The author
Pak. J. Pharm. Sci., Vol.26, No.1, January 2013, pp.217-232 217
Pharmaceutical and Industrial protein engineering
they are target of a host regulatory proteins (activators, manipulate different complicated processes such as
inhibitors, terminators and anti-terminators) that modulate regenerative medicine, tissue engineering, protein
gene expression (von-Hippel et al., 1984; Erie et al., delivery system, cell adhesion interaction modulation,
1992; Ikeda et al., 1986; Brautigam and Steitz. et al., signal transduction, understanding the protein-protein
1988; Freemont et al., 1988; Beese and Steitz, 1991). interface and analysis the protein 3D (Moss et al., 2009;
Costa et al., 2002; Liu et al., 2007; Holmwood and
Usage and importance Schindler 2009). Rational design has been improved by
There are many concepts behind improving the quality of modification in the site directed mutagenesis methods,
protein preparation for use in particular applications. In protein 2D and 3D, structure modelling, protein-protein
commercial applications, proteins can be less pure or interaction and their related software (Chie et al. 2004;
completely crude; however, they must be free from any Chie et al. 2005; Boeris et al. 2009; Kurgan 2008;
contamination in case of medicinal applications. Ludwig et al., 2003; Shen et al. 2007; Sugimoto et al.,
Pharmaceutical and medically used proteins should be 2004; Yan et al. 2008).
highly pure, active, correctly folded and biocompatible
(Degim and Çelebi, 2007; Woods and Hamuro 2001; The concept of protein 3D modeling is based on relating a
Nishijima 2005; Standley et al., 2008). Regarding the structure to the possible expected function(s) of amino
activity and stability, the protein must match perfectly the acids residues (homology modelling) and replacing them
reason of its usage. Although, the use of PE in industrial with other functions (molecular dynamics) in order to
applications has been increased significantly, screening of change their properties (Visegràdy et al., 2001). Rational
wild types organisms containing new proteins with design requires some knowledge about the basic protein
particular function(s) should not be stopped principle, genetic engineering tools and good skills in
(Satyanarayana et al., 2005). Organisms from extreme mathematics and algorithms and computing different
environments are becoming an important source of new software have been introduced to facilitate and reduce the
backbones for engineering proteins with significantly steps incorporated in the PE (Du et al. 2009; Quine et al.,
different properties and flourished the protein databases 2004).
with amazing structures (van den Burg, 2003). Successful
engineered proteins in general require proper combination Engineering protein is a sensitive and systematic process
of their properties. For example, protease used in Powder- that can get a breakdown if any mistake is committed. In
detergent would require stability against certain protein case of unwanted addition or loss of one or more base pair
stains. Properties, such as stability, high activity (in the during the sequencing process or stepwise analysis will
case of enzymes) and the ability to fold and interact lead to DNA frame shift, causing a change in its amino
correctly with surfaces are necessary for a variety of acids sequences. Important role of protein modelling was
industrially important proteins (Goodenough 1995; represented in designing a protein 3D structure from other
Runbingh 1997; Arulmuthu et al., 2009; Crisman and protein x-ray-structures (Chappell et al., 2002; Gaudier et
Randolph 2009). PE should be applied to solve al., 2002; Ozbek et al., 2002; Rhem et al., 2002; Vannini
complicated problems and introduce unexplored new et al., 2002; Woo et al., 2002; Akarsu et al., 2003;
properties (Chen et al., 2002; Fowler et al., 2002; Kan Calderone et al., 2003; Feinberg et al., 2003; Yun et al.,
2002; O'Maille et al., 2002; Best et al., 2003; Kiss et al., 2003; Ifuku et al., 2004; Parker et al., 2004; Timsit et al.,
2003; Nielsen et al., 2004b; O'Maille et al., 2004; Sueda 2006; Molina et al., 2009). Building a complete
et al., 2004; Tandang et al., 2005; Bai et al., 2007; Lin et hypothetical model for protein did not crystallized yet is
al., 2007; Alahuhta et al., 2008; Evdokimov et al., 2008). available by using the available protein structure database
(Christendat et al., 2002; Choi et al. 2009; Han et al.,
Manipulation of protein through gene 2005; Hattori et al., 2005; Holmes et al., 2006; Han et al.,
Manipulation and modification of proteins chemically or 2008; Jang et al., 2009).
through its constituent of amino acids is a complicated
process, however modification of proteins through genes Different computational algorithms have been introduced
has been proved as an easier and more efficient approach. to identify the amino acid sequences that have low
The basic techniques of genetic engineering are altering energies for target structures. The sequence-conformation
the responsible genes and generate the proteins with novel space under investigation is usually large. Software for
activities or properties. Such manipulations have proteins modeling has been established with different
frequently been used to discover structure/function(s) ability to detect the presence of un-natural molecules
relationships, as well as to alter the activity, stability and (Anfinsen, 1972; Briki et al., 2002; Dana et al., 2006;
properties of proteins (Gan et al., 2002; Milik et al., Chen et al., 2007; Cavasotto et al., 2008; Bordoli et al.,
2003). 2009; Chen et al., 2009; Chen and Shi 2009).
Rational design-the logical approach Some of the software is able to display and manipulate the
Rational design plays a crucial role in PE Phenomena and protein 3D structures (defined by X-ray analysis).
218 Pak. J. Pharm. Sci., Vol.26, No.1, January 2013, pp.217-232
Continues development of different software, instruments, design of new larger protein is restricted to redesigning of
x-ray machines, synchrotrons etc., together with the existing ones, building new proteins is successfully
advancement in the field of molecular biology and genetic achieved especially for their active sites. The re-design of
engineering brought tremendous progress in the fields of the hydrophobic core has been performed on number of
PE (Fieulaine et al., 2001; Thore et al., 2003; Ball, 2008; proteins, including the four-helix bundle ROP (Munson et
Herrmann et al., 2002a; Herrmann et al., 2002b; al., 1994) and ubiquitin (Munson et al., 1994; Lazar et al.,
Calderone 2004; Yao et al., 2006; Fukunishi and 1997).
Nakamura 2008; Abendroth et al.,; Procopiou et al., 2004;
Sugimoto et al. 2004; Yu et al., 2004; Nicolini and Chemical synthesis
Pechkova 2006; Pechkova and Nicolini 2006; Yu et al., Peptide synthesis can be produced chemically using
2009). organic synthesis techniques (Guzmán et al., 2006; Bang
et al., 2005). Chemical ligation is used to produce
Directed evolution peptides in high yield. The use of different strategies for
Directed evolution is an approach concerning with chemical synthesis allows the researcher to introduce un-
changes in the existing genetic material to modify its natural amino acids into the protein polypeptide chains,
structure/function(s) (Yuen and Liu 2007). Natural such as attachment of fluorescent probes to amino acid
evolution occurs under different pressures conditions but side chains. However, these methods can only be used on
with capacity to save the organism from the newly small scales (such as in the laboratory) but not on
activated pressure. Directed evolution has been developed commercial scale (Bray et al., 2003). Chemical synthesis
using controlled or designed selection to introduce new has been determined inefficient for polypeptides having
selected function(s). It can be obtained through the design more longer than 300 amino acids (Mirzaei et al., 2008).
of successful selection protocol for the newly expected The synthesized proteins may not readily assume their
mutants. native tertiary structure (Finkelstein and Galzitskaya et
al., 2004). Most chemical synthesis methods proceed
The directed evolution is widely used to solve different from C-terminus to N-terminus, which is almost opposite
industrial problems and to improve protein properties. It to the biological reaction (Ostermeier 1999; Schmidt et
has also been cited in terms of molecular evolution, al., 2001). However, chemical synthesis of protein is
sexual PCR and in vitro/vivo evolution (Zhao et al., highly important, especially on the peptide level, while
1997). It is the technique of preparing protein variants by manipulating protein through gene is easier (in most
recombining gene fragments in vitro, using PCR (Ko and cases). Meanwhile, manipulating short peptide through
Ma, 2005). After gene modification the new expressed chemical synthesis is of great importance especially in
protein can be subjected to another cycle(s) of pharmaceutical applications (Qabar et al., 1996).
modification (Crameri et al., 1998).
Mutagenesis
de novo design of proteins [protein from scratch] Change of amino acid(s) in a particular protein could
Enhancement of knowledge about different protein happen naturally and lead in many cases to sever human
structure/functions encourage scientist to design proteins diseases (Kao et al., 2000). Chemical mutagenesis is an
de novo (Iwata et al., 2001; Dai et al., 2002; Matsuura old method used for long time in strain improvement but
and Pluckthun 2004; Khodagholi et al. 2008; Klepeis et in most cases it did not deliver where the mutation(s) has
al., 2005; Takekiyo et al., 2007; Zou et al., 2007; Wang et been happened (Chiang, 2004). The mutated DNA
al., 2008; Shiga et al., 2009). They used information sequence using chemical mutagenesis is usually unknown
about the molecular recognition, conformational (Chiang, 2004). Mutant induced chemically or by
preferences, and structure analyses of native proteins radiation can happen in any gene(s) (Ermakova-Gerdes et
together with latest software for data analysis and al., 1996). It is the role of the selection process to select
manipulations. The design of new proteins is based on the the correct mutant (Michel et al., 2000). Moreover, it is
available information, especially those about protein 3D important here to state that the mutagenesis should be
structure. Any kind of knowledge about 2D and 3D wisely used. First priority will be for isolating of different
protein structure will be of great important (Bryson et al., kinds of proteins from nature. However, in certain cases
1998). One example about the newly designed protein is particular protein needs to be engineered (Baum et al.,
α-helical peptides that catalyze helical peptide ligation, 2004). It is important to direct the power of PE correctly
and was first reported by Ghadiri and co-workers (Severin and the screening of the existing natural protein(s) should
et al., 1997). The positioning of the peptide substrate for not be stopped and should continue on priority basis.
catalysis is due to hydrophobic and electrostatic
interactions between the two helices (Scheraga, 1985). Site directed mutagenesis
Peptide ligation depends on the reaction between an N- "The first step from the filed of genetic engineering to
terminal cysteine and a C-terminal thioester to form an protein engineering"
amide bond between two peptide entities. Although, the The use of PE started in 1978 when a site directed
mutagenesis was introduced in the laboratory of Michael and Maloy 2006; King et al., 2006; Ellrott et al., 2007;
Smith (Hutchison et al., 1978). Michael Smith and his co- Jiang and Blouin 2007; Tang et al., 2007; Thaa et al.,
workers were the first to introduce synthetic 2008).
oligonucleotides mediate mutations (Hutchison et al.,
1978). The first modification of an enzyme, a tyrosyl- Hybrid/fusion proteins
transfer RNA synthetase, using these tools was performed Protein sequences can be joined or fused by another
in 1982 (Winter et al., 1982). Through site-directed protein. The resulting protein is then called a hybrid,
mutagenesis a cysteine was replaced by a serine altering fusion, or chimeric, which generally has characteristics of
the protein’s substrate binding characteristics. His the joint proteins. Protein fusions have been extensively
revolutionary work within this field earned Michael Smith used to study an interaction between two or more proteins
the Nobel Prize in 1993. Many alterations in a particular (Kang and Jang 2009; Rehm et al., 2002; Ludwig et al.,
protein can be generated by making amino acid 2003).
replacements at specific site in the polypeptide backbone.
Each protein is unique in its amino acids composition. At Random mutagenesis
any position in the sequence, an amino acid can be The probability that need to change particular protein is
replaced by another using site directed mutagenesis very high. Proteins, which are compose of 20 different
method (Flaman et al., 2001; Bennett et al., 2003; Ho et amino acids, need significant number of site directed
al., 2005; Kasrayan et al., 2007; Choi et al., 2009; mutagenesis steps to alter its structure/functions. Random
Edelheit et al., 2009). As an example, Pancreatic mutagenesis become a best choice when there is a
ribonuclease A is an enzyme comprising 124 amino acids, shortage of existing information about protein
which cleaves the covalent bonds by the joining of structural/function(s) and the role of active residues
ribonucleic acids (RNA). If at position 119 in the (Amara et al., 2001, 2002; Amara, 2003). Random
sequence the naturally occurring histidine is replaced by mutagenesis is applied to a gene coded for a protein. The
an alanine, this mutant protein is expected to have little or process passes through a selection protocol(s) that enable
no biological activity, because histidine 119 is important to select newly mutated genes by detecting a difference in
for that activity. Other mutations have very little effect on the expressed protein behaviours after the mutagenesis
their proteins. This is particularly true when the amino steps. Genes could be a subject of more than one cycle of
acid is substituted by other closely related amino acids mutagenesis (Greener et al., 1997). Random mutagenesis
and when the amino acid is not conserved in the same usually seems to be time consuming process, but
protein found in other organisms (Branningan and alternative methods will be impossible if there is
Wilkinson 2002; Lippincott-Schwartz and Patterson 2003; insufficient knowledge about protein structure/functions.
Wang and Schultz 2002). Another example about site Random mutagenesis mimics what could happen naturally
directed mutagenesis will be discussed in the following that satisfies all the criteria leading to mutate certain gene;
sections of the review. even randomly but with more force and directed strategy.
Random mutagenesis can be used either in vivo using
Deletion and Insertion mutants mutator strains or in vitro using different kinds of PCR
Different amino acid can be deleted from the sequence, random gene modification protocols. As an example a
either separately or in groups (Chen et al., 2003). These detailed in vivo random mutagenesis protocol for mutated
processes are referred to as deletion mutants. Deletion phaCAp synthase gene has been described by Amara et al.,
mutation is usually used to map important region in a 2002. The protocol has given a direction to select mutates
particular protein. Or, to reduce the protein size, by of PhaCAp synthase with enhanced activities, change the
deleting apparently unnecessary part of a specific substrate specificity and give polymers with different
function. This usually results in missing one or more monomeric composition. The in vivo random mutagenesis
functions or properties of the wild type protein. It has is based on mutating strain E. coli XL1 Red (Stratagene©)
been proved useful in creating smaller proteins that match that is impaired in three of the DNA repairing mechanism
perfectly the demand of different applications and is used pathways (Glickman and Radman, 1980).
to understand the importance of each part of the protein
(Tratschin et al., 1984). Interestingly the changed amino acids were not in the
active site, which indicate a clear role of the
Whenever the deleted part impaired the protein function, structure/function(s) relationship over the enzyme active
that mean the protein active sites have been either sites as reported by Amara et al., (2001, 2002). Taguchi et
partially or completely deleted (Liu et al., 2000). Deletion al., (2001, 2002) used another strategy based on in vitro
mutation has been used to get rid of certain properties; in mutagenesis for phaCRe using PCR. It is important to
contrast, insertions mutation deal with inserting different highlight that a successful selection method should be
amino acid relate base pairs within the gene sequence used to efficiently recover the mutated gene from the wild
(Vorberg et al., 2001; Fernando et al., 2002; Ermakova et type gene.
al., 2003; Nielsen et al., 2004a; Yang et al., 2005; Brown
DNA shuffling activity and stability within a broad range of pH and

The DNA shuffling technique, introduced by Stemmer temperatures, and should be compatible with various
(1994), sets the directed mutagenesis approach apart from detergent components along with oxidizing and
the earlier random mutagenesis, which enable to combine sequestering agents (Horikoshi 1999; Ito et al., 1998). PE
different DNA fragment from different proteins variants has been used to improve the stability of BPN’ from
randomly using PCR. The screening efforts of Crameri et Bacillus amyfoliquefaciens in the chelating environment
al., (1998) have successfully applied this approach to of the detergent by deleting strong calcium-binding site
improve the whole cell fluorescence by green fluorescent (residues 75-83) and re-stabilizing the enzyme through
protein, which is widely used as a reporter for gene interactions without involving the metal-ion binding.
expression and regulation (Stemmer 1994; Crameri et al., Stability increase of more than 1000-fold in 10 mM
1998). EDTA have been reported for this protease (Strausberg et
al., 1995).
You and Arnold (1994) in their study show a 471-fold
increase in the activity of subtilisin E in 60% aqueous The surface properties of BPN’ have also been
dimethylformamide. The engineered enzyme can function engineered. Variants of mutates produce negative charges
in organic solvents, which is different from its natural in the active site region of the molecule adsorbed less
environments (You and Arnold, 1994). strongly (Broda et al., 1996) and gave better laundry
performance (Brode et al., 1966; Rubingh, 1996a, b).
Tailoring fundamental properties Cold-adapted maturation of thermophilic WF146 protease
Stability, activity, and surface properties are the most by mimicking the propeptide binding interactions of
important criteria of technical enzymes. Isolation of Psychrophilic subtilisin S41 has been designed by Yang
enzymes with extra properties like extremophilic enzymes et al (2008).
can be used to satisfy some of the PE demands that reduce
the amount of engineered protein (Schäfer et al., 2005; Pharmaceutical applications
Leuschner and Antranikan 1995; Gomes et al., 2004). By rough estimate, about 350 biotechnology drugs
currently undergo development. These include vaccines,
PE Applications gene therapy, antisense technology, and antibodies
It increases an understanding about the enzyme derived from ‘humanised’ transgenic mice (Whittingham
properties, such as stability, activity, surface properties, et al., 1997; Muller et al., 2004).
purification and introduction of new applications.
PE has been used to produce therapeutic pharmaceutical
Technical enzymes proteins with improved properties such as increased
In contrast to other fields of science, research which solubility and stability.
attracts the attention of industrial sectors is concerned
with both cost and profit. Major protein based drugs applications problem
Different proteins which show activity in vitro and have a
Technical enzymes are one of the major biotechnological promising role in medicinal applications have been filed.
industrial products, which had a market share of about 1 The reason is that they are primary molecules with
billion USD in 1999 (Schäfer et al., 2005). Part of these suboptimal affinity and poor half-life in vivo, which lead
products is the thermostable enzymes, which are well to poor efficacy (Moore et al., 1993). In other cases, many
consumed in different industrial processes and constitute of the original protein drug molecules are nonhuman that
more than 65% of the worldwide market (Leuschner and caused immune responses against the drug itself. Affinity,
Antranikan, 1995; Rao et al., 1998). Enzymes have been half-life, and dosing regime are all inter-related and play
used in many important industrial products. Their their role in determining the clinical efficacy and financial
applications can be made in paper industry, detergent, viability of protein-based drugs. This enhanced
drugs, degradation of different wastes, textile, food, understanding about the issues affecting a success in drug
pharmaceutical, leather, degumming of silk goods, development has been augmented by increased
manufactureing of liquid glue, cosmetics, meat capabilities in PE and selection/screening technologies.
tenderization, cheese production, growth promoters etc These technologies have been used to improve the
(Leuschner and Antranikan, 1995; Rao et al., 1998; effectiveness of a number of proteins used as drug (Ki et
Gomes and Steiner, 2004; Cowan, 1996). Proteases have al., 2003).
been used in many industrial processes including
detergent, wool quality improvement, meat tenderization, Reducing the immunogenicity of protein drug
leather, etc, (Amara and Serour 2008; Gupta et al., 2002a; molecules
Gupta et al., 2002b; Poza et al., 2007; Tang et al., 2004; Different non-human proteins, which appear in vitro high
Thangam and Rajkumar 2002; Valer, 1975). Ideally, the activity, are bonded by the immune system. This lead to
proteases used in detergent formulations should have high focusing on using protein from human sources/or
humanized protein (Conner et al., 1998; Shen et al., Polyketide synthases

2006). Antibiotics such as erythromycin are made by large
multidomain proteins called polyketide synthases
For example Pulmozyme (Genentech) is human DNAse (Findlow et al., 2003; Chin et al., 2006; Lai et al., 2006;
derived drugs used in managing cystic fibrosis and bovine Alekseyev et al., 2007). Site-directed mutagenesis has
pancreatic DNAse I (Shak et al., 1990). The been used to modify the substrate specificity of the
immunoginicity of mouse antibodies in human protein polyketide synthase reaction so that the new product
was one of the major problems of the early monoclonal contains a malonate unit, whereas the product of the
antibodies. Chimaeric antibodies by fusing mouse original enzyme contained a methylmalonate unit
variable domains to human constant domains improve the (Crawford et al., 2006). In addition to site-directed
body acceptance. This chimaeric retain binding specificity mutagenesis, the order of the polyketide synthase domains
and reduce the amount of mouse sequence in their has been shuffled to create proteins that could catalyze the
backbone. synthesis of new antibiotics (Branningan and Wilkinson
2002).
In 1998, Remicade (Centocor), a TNFa-neutralising
chimaeric monoclonal antibody, was approved for use in From basic research to products
treating Crohn’s disease and rheumatoid arthritis It is important to direct basic research to introduce certain
(Breedveld, 2000). A reduction in monoclonal antibody products. In fact, the science and market are tightly
immunogenicity has taken a stage further by related to each other by many aspects. Previously a
complementarily-determining region (CDR) grafting, description about industrial (e.g. Protein) and
where the CDRs of mouse antibodies were grafted onto Pharmaceutical approach (eg. Catalytic antibodies) have
human frameworks to further reduce the proportion of been presented which have direct application in the
mouse sequences in the drug while retaining its binding Market. However, there are many other examples about
specificity (Jones, 1986). basic research, which have been converted to commercial
products (Crawford et al., 2006).
Examples
Insulin From tem-1 β-lactamase to GeneEditorTM (Promiga©)
Insulin (lispro and aspart) was engineered through "Apparently, engineering antibiotic resistant gene is not a
mutagenesis to create monomeric forms, which are fast good idea, however the scientist imagination and
acting (Sensh et al., 2010). Conversely, another form of innovation enable them to see behind that"
insulin (glargine) was created by mutagenesis to β-lactamase is the resistant factor to the β-lactam
precipitate upon injection and give a sustained release of antibiotics. The plasmid-encoded TEM β-lactamase is the
insulin. Whittingham et al., (1997) have reported a crystal most prevalent one in Gram-negative enteric bacteria
structure of prolonged-acting insulin with albumin- (Venkatachalam et al., 1994; Matagne 1998).
binding properties (Sanger and Thompson 1953a,b;
Skelton et al., 2001; Vajdos et al., 2001; Boes et al., A discovery of new generation β-lactam antibiotics leads
2002; Gavira et al., 2002; Teixeira et al., 2002; Weiss et to induce new resistant strains that are reported as
al., 2002; Miyahara et al., 2003; Headey et al., 2004; Hua sensitive. The scientists are typing to understand why
and Weiss 2004; Fortier et al., 2005; Li et al., 2005; Mark microbes have the ability of conversion to a newly
et al., 2005; Sala et al., 2005; Huang et al., 2006; Kuang resistant form.
et al., 2006; Chandrashekaran et al., 2007; Kim et al.,
2007; Wan et al., 2008). Venkatachalam et al., (1994) have investigated TEM β-
lactamase variants with amino acid substitutions in the
Catalytic antibody active-site pocket of the enzyme. The experiments have
Antibodies are proteins that normally bind to a specific been identified in natural isolates with increased
molecule but do not alter the bound molecule in any way resistance to extended-spectrum cephalosporins, such as
(Coenraad et al., 2005). A catalytic antibody is a variant cefotaxime and ceftazidime. Mutants were selected for
of an antibody which has been changed by mutations to 100-fold more ceftazidime resistance than wild-type. All
have a novel sequence that folds into a structure, resulting mutants had a serine substitution at position 238, a lysine
into a specific reaction (such as amide bond formation, or arginine at position 240, and a small amino acid at
ester hydrolysis, and decarboxylation). Catalytic position 241. The role of each substitution was
antibodies function like enzymes, and are created to investigated by constructing individual G238S, E240K,
catalyze reactions for which there are no naturally and R241G mutants as well as the G238, SE240K double
occurring enzymes (Paul et al., 1994; Ali et al., 2009). mutant. The G238S mutant increases catalytic efficiency
Fifty or more reactions have been made by the action of for both ceftazidime and cefotaxime. However, to achieve
catalytic antibodies, which were obtained individually by significant increases in catalytic efficiency, both G238S
the methods of PE (Branningan and Wilkinson 2002). and the E240K mutants are required. The R241G mutant
results in a small increase in catalytic efficiency for only important, which can enhance its scientific and technical
ceftazidime. Using the same strategy the GeneEditor™ in applications. Scientists working on PE should be scaled,
vitro Site-Directed Mutagenesis System has been made. knowledgeable and patient. As an alternative, a scientific
This system uses antibiotic selection to obtain high group having scientists with different background can
frequency mutants. Selection of oligonucleotides provided lead to successful research findings. PE put great
the GeneEditor™ System encode mutations that alter the opportunities in the hands of scientists to improve the
ampicillin resistance gene, creating constructs that confer protein based industry within the frame of technical and
new additional resistance to the GeneEditorTM antibiotic pharmaceutical applications. The future brings an
selection mixture. Mutants generation using this system increasing interest in using PE and its applications to
retain ampicillin resistance and gain resistance to the industrial productions.
GeneEditorTM antibiotic selection mixture.
ACKNOWLEDGMENT
Several research studies have been conducted using
GeneEditorTM protocol. Amara and Rhem (2003) have This work was supported by Deanship of Scientific
described seven site directed mutations that have been Research and Research Centre, College of Pharmacy,
performed in PhaCPa, where 5 conserved residues were King Saud University, Riyadh, Saudi Arabia. I
replaced by site directed mutagenesis in order to identify acknowledge Assistant Prof. Dr. Haider Zaman from
the role of these amino acids in catalysis. Geophysics Department Faculty of Science, King Saud
University for some technical advices and revision of this
CONCLUSION AND FUTURE PROSPECTIVE review.
PE is a young discipline that has been branched out from REFERENCES

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Pharmaceutical and industrial protein engineering: Where we are?

Article in Pakistan journal of pharmaceutical sciences · January 2013

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Pharmaceutical and industrial protein engineering: Where we are?

Amro Abd-Al-Fattah Amara

Keywords: Protein engineering; applications; rational design; directed evolution.

Protein they were both awarded Nobel Prize in Chemistry in

*Corresponding author: e-mail: amroamara@web.de

DNA shuffling activity and stability within a broad range of pH and

humanized protein (Conner et al., 1998; Shen et al., Polyketide synthases

PE is a young discipline that has been branched out from REFERENCES

226 Pak. J. Pharm. Sci., Vol.26, No.1, January 2013, pp.217-232

Pak. J. Pharm. Sci., Vol.26, No.1, January 2013, pp.217-232 227

232 Pak. J. Pharm. Sci., Vol.26, No.1, January 2013, pp.217-232

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