The document discusses several examples of engineered proteins, including proteins engineered to treat cancer by visualizing and targeting it, and proteins engineered to create tools for research. It also discusses how protein engineering has been used to develop safer and more effective cancer therapeutics by customizing proteins. Several case studies are presented on engineering proteases to be oxidation-resistant for detergents, engineering tissue plasminogen activator as a thrombolytic agent, engineering insulins to mimic normal secretion patterns, and engineering post-translational modifications to target enzyme delivery.

1. Examples of engineered proteins,

2. • Protein engineered for visualizing and treating cancer
• Protein engineers are also using these powerful technologies to create
molecular toolkits for answering a wide range of research questions in
basic science, biotechnology, and biomedicine.
• In a number of human cancers, aberrant signaling through the Axl
receptor tyrosine kinase has been demonstrated to drive metastasis (Li et
al. 2009), confer therapeutic resistance (Hong et al. 2013), and promote
disease progression (Vajkoczy et al. 2006).
• Additionally, Axl overexpression has been observed in multiple solid and
hematological malignancies (Linger et al. 2008), with expression levels
often correlating with disease stage and poor clinical prognosis
(Gustafsson et al. 2009; Hong et al. 2013; Rankin et al. 2010).
• National Academies of Sciences, Engineering, and Medicine. 2017.
Frontiers of Engineering: Reports on Leading-Edge Engineering from the
2016 Symposium. Washington, DC: The National Academies Press.
://doi.org/10.17226/23659.

3. • AN ULTRA-HIGH AFFINITY ENGINEERED PROTEIN THERAPEUTIC
FOR TREATING METASTATIC DISEASE
• Research and development efforts over the past few decades have
culminated in a growing number of FDA-approved protein
therapeutics that enable targeted treatment of cancer.
• In parallel, continued efforts to develop safer and more effective
cancer therapeutics are being fueled by expanding knowledge of
mechanisms underlying disease pathophysiology and the ability to
customize proteins using a variety of engineering methods.
• Ref--National Academies of Sciences, Engineering, and Medicine.
2017. Frontiers of Engineering: Reports on Leading-Edge
Engineering from the 2016 Symposium. Washington, DC: The
National Academies Press. https://doi.org/10.17226/23659.

4. • Identifying naturally supercharged proteins
• One concern of using engineered proteins such as + 36 GFP for in
vivo applications is that the protein may provoke an immune system
response.
• To identify naturally occurring supercharged human proteins that may be
less immunogenic than engineered supercharged proteins, and sorted the
human proteome to identify the most positively charged proteins.
• These proteins are also able to enetrate mammalian cells and functionally
deliver associated macromolecules in vitro and in vivo.
• The naturally supercharged human protein (NSHP) Python script that we
use to identify NSHPs from the PDB and Swissprot databases will be
provided upon request.
• The following is a protocol for identifying supercharged proteins from
collection of protein sequences:

5. • CASE STUDY 1: OXIDATION-RESISTANT PROTEASES
• A range of enzymes are now added to clothing
detergents to help remove biological-based dirt such as
food and blood.
• In addition to proteases, these include lipases,
amylases, and cellulases.
• CASE STUDY 2: THE ENGINEERING OF TPA
• Tissue plasminogen activator (tPA)1 is a 527-amino
acid, 70-kDa serine protease produced by vascular
endothelial cells.
• It triggers activation of the fibrinolytic (clot-degrading)
system via proteolytic activation of plasminogen (Fig.
3) and thus has found application as a thrombolytic
agent in the treatment of heart attacks and strokes.

6. FIG. 3. The fibrinolytic system. tPA proteolytically converts the protease zymogen
plasminogen into plasmin. Plasmin, an active protease, in turn proteolytically
degrades fibrin molecules, which forms the structural basis of a blood clot.
Both tPA and plasminogen have fibrin binding sites, ensuring that activation of this
system occurs efficiently and selectively at the clot surface.

7. • CASE STUDIES 3 AND 4: ENGINEERED INSULINS
• The World Health Organization estimates that some 170million
people worldwide suffer from diabetes mellitus.
• Although only a minority of these suffer from insulin-dependant
(type 1) diabetes, world demand for purified insulin approached 5
metric tons in 2000 [14] and
•  is continually increasing.
• Healthy humans secrete insulin continuously at a low basal level,
with rapid but transient increases triggered by elevated blood
glucose concentrations.
• An Engineered Slow Acting Insulin—In addition to fast acting
insulins, slow acting insulin must usually be administered to
diabetics to mimic ongoing basal insulin secretion characteristic of
the normal state.

8. • CASE STUDY 5: ENGINEERING POST-TRANSLATIONAL
• MODIFICATIONS
• A small number of commercialized therapeutic
proteins are subject to post-translational engineering
to achieve a specific therapeutic goal.
• Gaucher disease is a relatively rare genetic disease
triggered by a lack of lysosomal glucocerebrosidase
activity [20].
• Glucocerebrosidase catalyzes the degradation of
glucocerebroside (a glycolipid), forming glucose and
ceramide.

9. FIG. 4. A simplified overview of the post-translational engineering
of the carbohydrate component of glucocerebrosidase to target its uptake
specifically to macrophages.
A more detailed treatment is available in Ref. [21]. M, mannose; SA, sialic acid.

12. • Protein engineering is an area possessing initiation hot
spots in recombinant DNA technology, where
managements in gene are expressed as alterations in
protein conformation responsible for desired properties.
• Various techniques for the specific engineering proteins can
principally be classified as techniques, which require
comprehensive prior acquaintance of protein, founding the
concept of rational technique of directed evolution that
aids in expression of the progression of natural evolution.
• Protein engineering so far has been flourishing to produce
proteins, which have rewarding applications in industry,
health and medicinal sciences, and ultimately in
nanobiotechnology in current scenario.