roebe 0.5.191

data/lib/roebe/www/protein_engineering/protein_engineering.rb

@@ -0,0 +1,725 @@
+#!/usr/bin/ruby -w
+# Encoding: UTF-8
+# frozen_string_literal: true
+# =========================================================================== #
+# http://localhost/programming/ruby/src/roebe/lib/roebe/www/protein_engineering/protein_engineering.cgi
+# =========================================================================== #
+require 'cyberweb/autoinclude'
+
+english('Protein Engineering') {
+created_on '01.05.2022' # Sunday.
+autoextend 
+ruby_favicon
+default_template ''
+body_css_class 'mar0px s2px padt2px VERDANAs'
+body_css_style 'background-color: #d3d2d1;'
+default_font_size_and_hyperlinks
+
+doc_preferred_width('mar0px pad0px s0_8em') {
+  h1 dot(106, 'marr8px')+
+     'Protein Engineering',
+     'mart1px marb0_5em'
+  # ========================================================================= #
+  # === A short history of protein engineering
+  # ========================================================================= #
+  fancy2 'A short history of protein engineering'
+  p_default_le {
+    e '<b>Protein engineering</b> would not have been possible without
+    techniques and advances originating from <b>molecular biology</b>,
+    such as recombinant DNA technology.'
+    br
+    e 'These techniques scientists to precisely substitute specific
+    amino acid residues, also known as <b>rational protein
+    modification</b>. This allowed scientists to reason about
+    individual amino acids in an enzyme, for instance.'
+    br
+    e 'In <b>1985</b>, Estell and coworkers modified <b>subtilisin</b> by
+    rational protein engineering, replacing Met222, which had been
+    identified as a site sensitive to oxidative damage. In this
+    work, the team was largely guided by crystallographic
+    information that was already available about <b>subtilisin</b>.'
+    br
+    e "While that early study from 1985 in regards to subtilisin
+    was a success, many other rational engineering attempts
+    failed - a reflection of a lack of understanding in regards to
+    of enzymes' true structural and functional complexity."
+    br
+    e '<b>DNA shuffling</b> was introduced by Stemmer and coworkers, to
+    offer an effective approach to address the shortcomings of
+    <b>random mutagenesis</b>.'
+  }
+  # ========================================================================= #
+  # === What are the prerequisites for the high-throughput screening
+  #     of "cells as bioreactors"?
+  # ========================================================================= #
+  fancy2 'What are the prerequisites for the high-throughput screening of
+  "cells as bioreactors"?'
+  p_default_le {
+    le 'Researchers seek to <b>create recombinant protein variants</b>
+    that are then <b>screened and evaluated</b>.'
+    br
+    le 'Manual steps are cumbersome, though, so efficient
+    <b>high-throughput (HTP) screening</b> approaches are desired,
+    in order to <b>improve the efficiency in early process
+    development</b>.'
+    br
+    le 'Different systems exist with different characteristics.
+    For instance, the <b>BioLector micro-bioreactor</b>
+    (<b>µ-bioreactor</b>) system can be used to screen
+    <i>E. coli</i> expression clones. The screening process
+    plays an important role in identifying diverse and
+    predictably productive cell lines for evaluation in
+    bioreactors.'
+    br
+    le 'The desired objective is to gain insight into biopharmaceutical
+    applications.'
+    br
+    e '<b>High throughput screening</b> (<b>HTS</b>) is the use of
+    <b>automated equipment</b> to rapidly test thousands to millions of
+    samples for biological activity at the model organism, cellular,
+    pathway, or molecular level. HTS typically aims to screen
+    100000 or more samples per day. Thus, one <b>prerequisite</b>
+    is that the procecure is relatively simple and ideally
+    makes use of automation-compatible assay designs, under
+    assistance by a robot, such as for sample handling.
+    Automated data processing is also crucial.'
+    br
+    e 'HTS is commonly used in pharmaceutical and biotechnology
+    companies to identify compounds with pharmacological or
+    biological activity. These compounds are then called 
+    <b>hits</b>.'
+    br
+    e 'Typically, HTS assays are performed in microtiter plates
+    in 96-, 384-, or 1536-well formats while traditional HTS
+    usually tests each compound in a compound library at a
+    single concentration, most commonly 10 μM.
+    The 384-well plate format is now standard for HTS, with
+    an increasing number of screens using 1536-well plate
+    format or even higher well-density formats.'
+    br
+    e 'Another prerequisite may be to use cell sorting via
+    flow cytometry.'
+    br
+    e 'Sources used:'
+    br
+    selfy 'https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/high-throughput-screening',
+              css_class: 'mars3em BOLD' 
+  }
+  # ========================================================================= #
+  # === Fusion proteins
+  # ========================================================================= #
+  # http://localhost/programming/ruby/src/roebe/lib/roebe/www/protein_engineering/protein_engineering.cgi#fusion_proteins
+  # ========================================================================= #
+  fancy2 'Fusion proteins'
+  p_default_le {
+    e '<b>Fusion proteins</b> are created by <b>joining together different
+    proteins or different parts of proteins, yielding a novel molecule in
+    the process</b>. This is <b>a single polypeptide</b>.'
+    br
+    e 'Each of the different component parts of the fusion protein may
+    occur naturally as well.'
+    br
+    e 'An general example for a fusion protein may be <b>
+    <a href="https://www.sinobiological.com/resource/antibody-technical/chimeric-monoclonal-antibody">
+    chimeric antibodies</a></b>. These antibodies may have the Fab fragment
+    from one species fused with the Fc fragment from another. They were
+    engineered to make antibodies more human-like, in order to reduce
+    immune responses to these antibodies.'
+    br
+    e 'Chimeric antibodies based on mice antibodies are hybrids - the
+    VH area from the mouse is fused onto t he human CH1, CH2, CH3 part.
+    And the VL from the mouse is fused onto CL of humans. The nomenclature
+    used for chimeric antibodies is <b>-xi</b>.'
+    br
+    e 'Peptibodies consist of a protein component (the "pepti-" part)
+    and an antibody-like component (the "-body"). The antibody component,
+    usually the Fc part, increases the stability of the molecule as
+    a whole.'
+    br
+    e 'Recombinant fusion proteins can be modified in various parameters, such
+    as catalytic efficiency, <b>solubility</b>, <b>expression levels</b>,
+    <b>crystal quality</b> or <b>thermostability</b>.'
+    br
+    e 'Fusion tags may also help by acting as a nucleation site for the folding
+    of the target protein: the so-called molten globule hypothesis.'
+  }
+  # ========================================================================= #
+  # === Applications of Fusion Proteins
+  # ========================================================================= #
+  # http://localhost/programming/ruby/src/roebe/lib/roebe/www/protein_engineering/protein_engineering.cgi#applications_of_fusion_proteins
+  # ========================================================================= #
+  fancy2 'Applications of Fusion Proteins'
+  p_default_le {
+    e 'The three most important use cases of fusion proteins are:'
+    br
+    reset_the_counter
+    counter 'reporters of expression level','mars3em BOLD darkblue'
+    counter 'aids in the purification of cloned genes','mars3em BOLD darkblue'
+    counter 'histochemical tag to enable visualization
+    of the location of proteins in a cell, tissue, or organism','mars3em BOLD darkblue'
+    br
+    e 'Fusion proteins can be used in research, as pointed out above, such
+    as as reporters of expression level. Two use cases will be described
+    next.'
+    br
+    e 'The first example that can be given is that we can attach fluorescent
+    tags to a protein in a host cell. This then allows the researcher to
+    track protein interactions in real time. The first fluorescent tag was
+    <b>GFP</b> (green fluorescent protein), and it is still in use today.
+    These are in general called <b>reporter genes</b> or <b>reporter
+    tags</b>.'
+    br
+    e 'The second example given for a fusion protein used in research is
+    a fusion protein used for <b>purification</b>, via affinity
+    chromatography. Examples here include staphylococcus protein A,
+    glutathione-S-transferase, 
+    <a href="https://en.wikipedia.org/wiki/Streptavidin">strepavidin</a>
+    or <b>polyhistidine</b>.'
+    br
+    e 'A third example can be given by <b>split GFP fusion partners</b>,
+    which are used for reporter constructs.'
+    br
+    e 'Fusion proteins can also be used in therapy - antibody-drug
+    conjugates may serve as an example. Two additional examples
+    will be described next.'
+    br
+    e 'The first example to be brought for fusion proteins in therapy
+    are Fc-fusion proteins that are composed of the Fc region of
+    IgG antibody (Hinge-CH2-CH3) and the desired, linked protein.
+    The Fc region of Fc-fusion proteins can bind to neonatal Fc
+    receptor (FcRn). The first therapeutic Fc-fusion protein
+    was introduced for the treatment of AIDS.'
+    br
+    e 'Another example for fusion proteins in therapy is
+    <a href="https://en.wikipedia.org/wiki/Etanercept">
+    <b>Etanercept</b></a>, which is used for the treatment of
+    autoimmune responses. Etanercept interferes with the
+    <b>tumor necrosis factor</b> (TNF). Etanercept is 
+    created by fusing the TNF receptor to the constant end of 
+    an IgG1 antibody.'
+    br
+    e 'Important parameters for the design of a fusion protein include:'
+    br
+    counter '- effective targeting and binding'
+    counter '- (lack of) cytotoxicity'
+    counter '- design of the linker; often an in-vivo cleavable
+    linker'
+    counter '- stable molecule with an extended half-life (the Fc
+    region of human IgG1 is generally chosen as the fusion partner
+    for the effector molecule(s) because it extends the fusion
+    protein half-life)'
+  }
+  # ========================================================================= #
+  # === Chimeric mutant proteins
+  # ========================================================================= #
+  # http://localhost/programming/ruby/src/roebe/lib/roebe/www/protein_engineering/protein_engineering.cgi#applications_of_fusion_proteins
+  # ========================================================================= #
+  fancy2 'Chimeric mutant proteins'
+  p_default_le {
+    e 'Chimeric mutant proteins may occur naturally due to genetic
+    mutations, such as:'
+    br
+    reset_the_counter
+    counter 'a chromosomal translocation','mars3em BOLD'
+    counter 'tandem duplication','mars3em BOLD'
+    counter 'retrotransposition','mars3em BOLD'
+    br
+    e 'This will create a novel coding sequence containing parts of the
+    coding sequences from two different genes.'
+    br
+    e 'Naturally occurring fusion proteins are commonly found in <b>cancer
+    cells</b>, where they may function as <b>oncoproteins</b>. The <b>bcr-abl fusion
+    protein</b> is a well-known example of an oncogenic fusion protein
+    and is considered to be the primary oncogenic driver of <b>chronic
+    myelogenous leukemia</b>. The name BCR is derived from
+    <b>Breakpoint Cluster Region</b>. Another example of a naturally
+    occurring fusion protein is the <b>Gag-onc fusion proteins</b>.'
+    br
+    e 'The most common way how this outcome may happen is when a 
+    chromosomal translocation replaces the terminal exon of one
+    gene with an intact exon from a second gene. This creates a
+    single gene that can be transcribed, spliced, and translated
+    to produce a functional fusion protein.'
+    br
+    e 'Many important cancer-promoting oncogenes are fusion genes
+    produced in this way.'
+    br
+    e 'Examples include:'
+    br
+    reset_the_counter
+    counter 'Gag-onc fusion protein','mars3em BOLD'
+    counter 'Bcr-abl fusion protein','mars3em BOLD'
+    counter 'Tpr-met fusion protein','mars3em BOLD'
+    br
+    e 'Antibodies are fusion proteins produced by <b>V(D)J recombination</b>.'
+    br
+    e 'Chimeric proteins may also be created via recombinant technology.'
+  }
+  # ========================================================================= #
+  # === Chimeragenesis methods
+  # ========================================================================= #
+  fancy2 'Chimeragenesis methods'
+  p_default_le {
+    e '<b>Chimeric proteins</b> are often created to <b>study structure-function
+    relationships</b>.'
+    br
+    e 'Generally speaking the techniques used to create chimeric proteins
+    can be divided into:'
+    br
+    e 'noncombinatorial approaches (also called <i>rational</i>)','BOLD mars3em'
+    e 'combinatorial approaches','BOLD mars3em'
+    br
+    e 'It is rather tedious to create a few hybrid proteins via the
+    rational approach, whereas the combinatorial methods can yield
+    more than 10⁸ (!) different hybrids in a single experiment.'
+  }
+  # ========================================================================= #
+  # === Protein linkers
+  # ========================================================================= #
+  fancy2 'Protein linkers'
+  p_default_le {
+    e 'Protein linkers aid fusion protein design by providing appropriate
+    spacing between domains.'
+    br
+    e 'Protein linkers permit important domain interactions, reinforce
+    stability, reduce steric hindrance and make them preferred for use
+    in fusion protein design even when N and C termini can be fused.'
+    br
+    e '<b>Flexible linkers</b> are typically made up of glycine and/or
+    serine, whereas <b>rigid linkers</b> are made up of proline.'
+  }
+  # ========================================================================= #
+  # === Affinity purification
+  # ========================================================================= #
+  fancy2 'Affinity purification'
+  p_default_le {
+    e 'Different tags have been used in affinity purification, including:'
+    br
+    reset_the_counter
+    counter 'staphylococcus protein A','mars3em BOLD'
+    counter 'glutathione-S-transferase','mars3em BOLD'
+    counter 'maltose-binding protein','mars3em BOLD'
+    counter 'cellulose-binding protein','mars3em BOLD'
+    counter 'chitin-binding domain','mars3em BOLD'
+    counter 'thioredoxin','mars3em BOLD'
+    counter 'streptavidin # but too strong as interaction','mars3em BOLD'
+    counter 'RNaseI','mars3em BOLD'
+    counter 'polyhistidine','mars3em BOLD'
+    counter 'antibody epitopes','mars3em BOLD'
+  }
+  # ========================================================================= #
+  # === Linker Peptides
+  # ========================================================================= #
+  fancy2 'Linker Peptides'
+  p_default_le {
+    e 'Linker peptides are often used when fusion proteins are created. Such
+    linker peptides act as spacers, and they ensure that the proteins will
+    <b>fold independently</b>.'
+  }
+  # ========================================================================= #
+  # === Spycatcher / SpyTag
+  # ========================================================================= #
+  fancy2 'Spycatcher / SpyTag'
+  p_default_le {
+    e 'The Spycatcher / SpyTag system was developed in <b>2012</b>.'
+    br
+    e 'The protein <b>SpyCatch</b> has a size of <b>12.3 kDa</b>.'
+    br
+    e '<b>FbaB</b> contains a domain with an isopeptide bond, between a
+    Lysine and an Aspartate.'
+    br
+    e 'A research group split this domain, rationally engineering the fragments
+    and obtained a peptide that they called <b>SpyTag</b>. This, in turn,
+    forms an amide bond to its protein partner called <b>SpyCatcher</b>.'
+    br
+    e '<b>SpyTag</b> (13 amino acids in length) has been designed starting
+    from the Streptococcus pyogenes fibronectin-binding protein 
+    called <b>FbaB</b>. The natural role of the <b>FbaB</b> protein is
+    to assist colonization of the host cell.'
+    br
+    e 'SpyTag can fairly easily be fused at either terminus,
+    or even internally.'
+    br
+    e 'The primary use case is that when we use the Tag/Catcher pair,
+    <b>bioconjugation</b> can be achieved <b>between two recombinant
+    proteins</b>.'
+    br
+    e '<b>SpyTag</b> is also known as "<i>an affinity detection tag
+    that binds forever</i>".'
+    br
+    selfy 'https://en.wikipedia.org/wiki/SpyCatcher'
+  }
+  # ========================================================================= #
+  # === Semirational protein engineering
+  # ========================================================================= #
+  fancy2 'Semirational protein engineering'
+  p_default_le {
+    e 'Semirational approaches capitalize on information from 
+    protein sequence and structure databases.'
+    br
+    e 'Additionally they may also benefit from machine-learning
+    algorithms, in order to guide the design of smaller, more
+    focused libraries of protein variants.'
+    br
+    e 'The simplest semirational approaches typically utilize
+    <b>multiple sequence alignments</b> to determine the degree
+    of evolutionary variability of amino acids at each
+    position in a protein sequence.'
+  }
+  # ========================================================================= #
+  # === General strategies for engineering new proteins
+  # ========================================================================= #
+  fancy2 'General strategies for engineering new proteins'
+  p_default_le {
+    e 'The two primary strategies used to engineer new proteins are:'
+    br
+    cmd '  (1) <b>diversity-oriented protein engineering</b> (also called
+    <b>directed evolution</b>'
+    cmd '  (2) <b>computational protein design</b>'
+    br
+    e '(1) Directed evolution identifies protein variants with
+    desired properties from a large pool of mutants. Its success
+    depends on <b>generating sufficient sequence diversity</b>.'
+    br
+    e '(2) Computational protein design generates and screens
+    protein sequences <i>in silico</i> before synthesizing them in
+    the laboratory.'
+  }
+  # ========================================================================= #
+  # === Directed evolution
+  # ========================================================================= #
+  # http://localhost/programming/ruby/src/roebe/lib/roebe/www/protein_engineering/protein_engineering.cgi#directed_evolution
+  # ========================================================================= #
+  fancy2 'Directed evolution'
+  dimg find_image(:directed_evolution),
+       'round_black3 mars3em'
+  p_default_le {
+    e 'Directed evolution <b>mimics</b> the process of evolution by introducing
+    random mutations into proteins, in the laboratory.'
+    br
+    e '<b>Directed Evolution</b> is an iterative process. A parental gene
+    sequence may be modified. Past that step, selection steps are used to
+    determine better variants, based on host cell fitness.'
+    br
+    e '<b>Directed Evolution</b> is also used in nature. For instance, the
+    rapid emergence and resistance to antibiotics is an example of directed
+    evolution, as well as the generation of metabolic pathways for degradation
+    of xenobiotics, such as pesticides.'
+  }
+  # ========================================================================= #
+  # === Using error-prone PCR to achieve diversity in proteins
+  #
+  # Diversifying a sequence via error-prone PCR introduces point mutations
+  # randomly. The diversity that can be achieved is, however had, somewhat
+  # limited. Why?
+  # ========================================================================= #
+  fancy2 'Using error-prone PCR to achieve diversity in proteins'
+  p_default_le {
+    e 'Assessing library diversity is an important control step in a
+    directed evolution experiment in the lab: "<i>Laboratory evolution</i>".'
+    br
+    e 'The fraction of proteins that retain wild-type function after
+    mutation has long been observed to <b>decline</b> exponentially as
+    the average number of mutations per gene increases.'
+    br
+    e 'Error-prone polymerase chain reactions (EP-PCR) is able to
+    generate libraries with up to 15-30 mutations per gene. If
+    one wishes to <b>introduce random mutations</b>
+    into a defined segment of DNA then <b>EP-PCR</b> is the
+    <b>method of choice</b>.'
+    br
+    e 'Diversifying a sequence via error-prone PCR introduces point
+    mutations randomly. The diversity that can be achieved is,
+    however, somewhat limited.'
+  }
+  # ========================================================================= #
+  # === Evolution of enzymes
+  # ========================================================================= #
+  # http://localhost/programming/ruby/src/roebe/lib/roebe/www/protein_engineering/protein_engineering.cgi#evolution_of_enzymes
+  # ========================================================================= #
+  fancy2 'Evolution of enzymes'
+  p_default_le {
+    e 'A gene coding for an enzyme encodes for a specific function.'
+    br
+    e 'These genes - and the function of enzymes - are under specific
+    selective pressure. Most mutations that happen naturally have a
+    negative effect, and are thus <b>not</b> beneficial.'
+    br
+    e '<b>Recombination</b>, <b>Mutation</b> and <b>Selection</b> contribute
+    primarily to changing gene sequences. In the lab we can use directed
+    evolution to change these gene sequences.'
+    br
+    e 'Average enzymes are far less efficient than "perfect enzymes".
+    The <b>average efficiencies</b> reflect the metabolic demand in the
+    cell. Thus, <b>K<sub>M</sub> values</b> correlate with cellular
+    metabolite concentration. <b>k<sub>cat</sub> values</b> also meet
+    metabolic flux demands.'
+    br
+    e 'A <b>perfect enzyme</b> makes no futile encounter with a substrate.'
+    br
+    e 'An <b>average enzyme</b> encounters 10_000 substrates and makes only
+    one conversion.'
+    br
+    e 'There is no evolutionary drive towards very fast enzymes. Enzymes
+    have to remain flexible in their structure - the "wobbling" effect
+    necessary for actual catalysis. Enzymes do not have to be very fast,
+    for the most part.'
+    br
+    e '<b>Evolved enzyme rates</b> have no correlation to how demanding
+    a reaction is.'
+    br
+    e '<b>Evolution</b> drives enzymes towards "good enough" rather
+    than perfect. In <i>E. coli</i>, the majority of enzymes have
+    potential for higher efficiency.'
+    br
+    e 'A faster enzyme may not necessarily be an improvement for
+    the target organism at hand, as the enzyme is working within
+    the context of a larger network of other enzymes and 
+    metabolites.'
+    br
+    e 'Evolving a new function in an enzyme will often come at a significant
+    cost for the primary function: such a new enzyme may be rather 
+    unstable.'
+  }
+  # ========================================================================= #
+  # === Library Selection
+  # ========================================================================= #
+  # http://localhost/programming/ruby/src/roebe/lib/roebe/www/protein_engineering/protein_engineering.cgi#library_selection
+  # ========================================================================= #
+  fancy2 'Library Selection'
+  p_default_le {
+    e 'A good slogan that can be applied onto Library Selection in
+    Protein engineering is:'
+    br
+    mars2em '„You Get What You Select For.“','italic'
+    br
+    e "An extremely important goal in library design is to link a 
+    library member’s genotype with its phenotype."
+  }
+  # ========================================================================= #
+  # === Cassette mutagenesis
+  # ========================================================================= #
+  fancy2 'Cassette mutagenesis'
+  p_default_le {
+    e 'In <b>cassette mutagenesis</b> restriction sites flanking the DNA
+    particular sequence to be replaced are digested with cognate
+    restriction enzymes and a replacement sequence is inserted between
+    these sites - see <i>Wells et al. 1985</i>.'
+  }
+  # ========================================================================= #
+  # === SUMO modification of proteins                              (SUMO tag)
+  # ========================================================================= #
+  # http://localhost/programming/ruby/src/roebe/lib/roebe/www/protein_engineering/protein_engineering.cgi#sumo_modification_of_proteins
+  # ========================================================================= #
+  fancy2 'SUMO modification of proteins'
+  p_default_le {
+    e '<b>deSUMOylating enzymes</b> reverse any prior SUMOylation.'
+    br
+    e 'SUMO modification of proteins has many functions:'
+    br
+    reset_the_counter
+    counter '- protein stability','mars3em BOLD'
+    counter '- nuclear-cytosolic transport','mars3em BOLD'
+    counter '- transcriptional regulation','mars3em BOLD'
+    br
+    e 'Typically, only a small fraction of a given protein is SUMOylated
+    and this modification is rapidly reversed by the action of deSUMOylating
+    enzymes. SUMOylation of target proteins has been shown to cause a
+    number of different outcomes including altered localization and
+    binding partners.'
+    br
+    e 'For instance, the SUMO-1 modification of RanGAP1 (the first
+    identified SUMO substrate) leads to its trafficking from cytosol
+    to nuclear pore complex.'
+    br
+    e 'The SUMO modification of hNinein leads to its movement from the
+    centrosome to the nucleus.'
+    br
+    e 'In many cases, SUMO modification of transcriptional regulators
+    correlates with <b>inhibition of transcription</b>.'
+    br
+    e 'SUMO has <b>very little sequence identity with ubiquitin</b>:
+    less than 20% at the amino acid level. Nonetheless it has a nearly
+    identical structural fold. The SUMO protein is a fairly small
+    <b>globular protein</b>: it contains only about 100 aminoacids and
+    has a weight of <b>12 kDa</b> only.'
+    br
+    e 'The SUMO protein has a unique N-terminal extension of 10-25
+    amino acids which other ubiquitin-like proteins do not have.
+    This N-terminal sequence is found related to the formation of
+    SUMO chains.'
+    br
+    e 'The SUMO tag is used in biotechnology in order to purify proteins
+    found in inclusion bodies in E. coli. Here the the protein of interest
+    is fused together with <b>a solubility tag</b> such as SUMO.
+    SUMO-tagged proteins in general fold more properly and it can
+    help when we face "difficult-to-express proteins".'
+    br
+    e '<b>SUMO</b> can be cleaved from the protein of interest using a 
+    <b>SUMO-specific protease</b> such as the <b>Ulp1 peptidase</b>.
+    Ulp1 peptidase was originally obtained from <i>yeast</i>.'
+  }
+  # ========================================================================= #
+  # === Solubility of proteins in Protein Engineering
+  # ========================================================================= #
+  # http://localhost/programming/ruby/src/roebe/lib/roebe/www/protein_engineering/protein_engineering.cgi#surface_display_techniques
+  # ========================================================================= #
+  fancy2 'Solubility of proteins in Protein Engineering'
+  p_default_le {
+    e 'A common challenge in fusion protein design is the issue of
+    insolubility of newly synthesized fusion proteins in the recombinant
+    host, leading to an over-aggregation of the target protein in the
+    cell.'
+    br
+    e 'Molecular chaperones that are able to aid in protein folding may
+    be added, thereby better segregating hydrophobic and hydrophilic
+    interactions in the solute to increase protein solubility.'
+  }
+  # ========================================================================= #
+  # === Surface Display techniques
+  # ========================================================================= #
+  # http://localhost/programming/ruby/src/roebe/lib/roebe/www/protein_engineering/protein_engineering.cgi#surface_display_techniques
+  # ========================================================================= #
+  fancy2 'Surface Display techniques'
+  p_default_le {
+    e 'An interesting strategy to circumvent membrane transport
+    limitations - and minimize host protein interference - has been
+    the development of <b>surface-display systems</b>.'
+  }
+  # ========================================================================= #
+  # === Phage Display
+  # ========================================================================= #
+  # http://localhost/programming/ruby/src/roebe/lib/roebe/www/protein_engineering/protein_engineering.cgi#phage_display
+  # ========================================================================= #
+  fancy2 'Phage Display'
+  p_default_le {
+    e 'In '+string_link(:phage_display, content: 'phage display',
+    css_class: 'BOLD')+', we can <b>study proteins and protein-protein
+    interactions on a combinatorial scale</b>, allowing the researcher
+    to <b>probe billions of different protein variants simultaneously</b>.'
+    br
+    e 'Phage display technology provides an in vitro version of
+    classical Darwinian evolution by establishing a physical
+    linkage between a polypeptide and the encoding genetic
+    information.'
+    br
+    e 'By mutating DNA encoding for a displayed polypeptide, it is
+    possible to generate a multitude of related variants. This
+    library of variants can then be displayed on the surfaces
+    of phage particles. Variants can be generated via a
+    <b>selection process</b>.'
+    br
+    e 'The usual property of interest is a specific binding activity.'
+    br
+    e 'Phage display was first developed with the
+    <i>Escherichia coli</i>-specific filamentous bacteriophage M13
+    in <b>1985</b> and is, in this case, more aptly called
+    <b class="darkblue">filamentous phage display</b>. The success of the
+    M13 phage display prompted the subsequent development of numerous
+    alternative display systems.'
+    br
+    e 'Although M13 was historically the first phage system
+    employed here, other phages were used as well, such as
+    the λ-phage in ~1998 and the <b>T4 phage</b>.'
+    br
+    e 'M13 is a filamentous bacteriophage with a ssDNA genome.
+    The virion has 65 Å in diameter, a length of 9300 Å and
+    contains about 6400 bases. Approximately 2700 copies
+    of the major coat protein (pVIII) can be found on its
+    surface.'
+    br
+    e 'The individual steps for phage display, at the least for the
+    <b>M13 display technique</b>, are as follows:'
+    br
+    cmd '  (1) generate a phage pool'
+    cmd '  (2) binding selection including the washing step (to
+    wash away non-binding phages)'
+    cmd '  (3) yield binding phages'
+    cmd '  (4) amplification step (in bacteria)'
+    br
+    e 'Positive clones are indentified by enzyme-linked
+    immunosorbent assay (<b>ELISA</b>).'
+    br
+    e 'The protein that is usually chosen for phage display
+    is <b>pIII</b>. This protein has a size of <b>42 kDa</b>
+    and consists of three distinct structural domains:
+    <b>N1</b>, <b>N2</b>, and <b>CT</b>. The <b>N2</b> domain
+    binds to the F-pilus of <i>E. coli</i>. The <b>N1 domain</b>
+    binds to the outer membrane protein <b>TolA</b>.'
+    br
+    e 'Once inside of E. coli the ssDNA viral genome is converted
+    into double-stranded DNA (dsDNA) by host proteins.
+    The task of the protein pV is to bind to the ssDNA genome of
+    M13.'
+    br
+    dimg 'science/viruses/M13_outer_structure.png',
+         'round_black3 mars4em'
+  }
+  # ========================================================================= #
+  # === Screening by selection
+  # ========================================================================= #
+  # http://localhost/programming/ruby/src/roebe/lib/roebe/www/protein_engineering/protein_engineering.cgi#screening_by_selection
+  # ========================================================================= #
+  fancy2 'Screening by selection'
+  p_default_le {
+    e '<b>Screening by selection</b> is an elegant and powerful approach
+    that establishes a connection between properties of the engineered
+    protein and the survival of an organism.'
+    br
+    e 'Here, <b>the engineered enzyme is vital for survival of the host
+    strain</b>. The applicability of this technique is limited, though.'
+    br
+    e 'This technique also often requires custom-made recipient strains,
+    in particular <b>deletion strains</b> for enzymes essential in
+    biosynthetic pathways.'
+    br
+    e 'A digital camera as well as image analysis software allowing the
+    identification and isolation of positives is often used in this
+    technique as well.'
+  }
+  # ========================================================================= #
+  # === Slogans                                                 (slogans tag)
+  # ========================================================================= #
+  h2 'Slogans'
+  p_default_le('mars2em') {
+    e 'Enzymes and antibodies are used for <i>in-vitro</i> diagnostic
+    devices.'
+    br
+    e 'Chemical synthesis can give access to complex proteins that
+    biological systems can hardly produce or cannot produce at all.'
+    br
+    e '<b>Chimeric proteins</b> are proteins that contain segments from two
+    or more different parent proteins.'
+    br
+    e '<b>Chimeragenesis</b> can be used as a tool for gaining insights
+    into enzyme mechanism.'
+    br
+    e 'Protein engineering makes it possible to <b>design</b> completely
+    new enzymes with specific properties for industrial applications.'
+    br
+    e 'Protein engineering requires identification of particular amino
+    acid sequences that will result in desired structural and
+    functional properties.'
+    br
+    e 'The rules defining <b>sequence-structure-function relationships</b>
+    are not well understood.'
+    br
+    e 'The complexity of proteins present engineering challenges whose
+    solutions will require a combination of experimental and
+    computational approaches.'
+    br
+    e 'Enzymes (which are proteins) are the key players of
+    <b>biocatalysis</b>.'
+    br
+    e 'Biocatalysts (such as enzymes) can be changed to
+    effectively operate in a desired reaction environment.'
+    br
+    e 'Inspiration for novel enzymes is often derived from
+    organic chemistry.'
+    br
+    e 'Protein engineering and directed evolution are powerful
+    technologies for probing protein sequence-function
+    relationships.'
+  }
+  br
+}}