Introduction

Collagen protein

All extracellular matrices are primarily composed of collagenous proteins. Collagen has

traditionally been viewed as having a structural role to play. However, it has become evident

in the last decade that collagen is a heterogeneous class of molecules, many of them

possessing the structural properties classically related to "collagen," but many others

possessing various other properties. According to Abbott et al. (2021), collagen fibers are

made up of monomeric building blocks called tropocollagens. A collagen molecule contains

three polypeptide chains composed of two alpha-chains that are identical, as well as a

separate alpha-2 chain, arranged in a triple helix with coiled coils. Gly-Xaa-Yaa triplets

constitute most of this protein's structure, with Pro at location Xaa and OH-Pro at position

Yaa. The translation product must undergo an extraordinary amount of posttranslational

processing to mature into fibrillar material with great strength, such as found in collagen-rich

tissues such as skin and tendon. In the vertebrates, collagen exists in 15 different forms. Their

distribution is tissue-specific, they occur at defined times and places during development, and

they have different properties (Veit et al., 2006). Furthermore, collagens have been

demonstrated to be involved in several cellular processes, either directly or indirectly,

including cell attachment and differentiation, chemotaxis, antigens in immunopathology, and

the defective component in various pathologies (Merrett, Wan, Lee, Harden, & Engineering,

2021). Furthermore, collagens may play numerous physiological and developmental roles,

which remain to be identified.  Different animals have different forms of collagen. The type I

and type III collagen may be found in cows. Collagen is essential for the moisture and

elasticity of the skin. Marine tissues include Type I and Type II collagen. Although marine

collagen has not been well studied, it has the potential to improve the health of skin and

cartilage. Marine collagen's advantages are still being studied, but they may include UV
protection, young skin, and healing. Type II collagen is found in chicken collagen, as well as

Types I and V collagen from eggshells. Chicken collagen may help joints, cartilage, and

ligaments by promoting the body's reaction to inflammation. Collagen makes up round about

30% of total protein present in the body, with cartilage accounting for 60%. Collagen

synthesis starts to decrease at the age of 30, resulting in skin that is thinner, drier, and less

elastic. However, Collagen supplements are available on the market if you're concerned about

collagen loss (Lyng, Cai, & Bedane, 2022).

Types of collagen protein:

Type I Collagen

The majority of the organic matrix of the bone is composed of type I collagen, which

accounts for 90% of it. Because of its vast spectrum of advantages, type I collagen is found in

most supplements (Naomi, Ridzuan, & Bahari, 2021).

Bovine, marine, and eggshell collagen is all sources of type I collagen. Type I collagen may

contribute in the formation of mineral crystals in the bone. Despite the need for greater

research, many consumers prefer collagen Type I to keep their hair, nails, and skin in good

shape. The endoplasmic reticulum produces type I collagen by combining pro-collagen alpha-

1 (COL1) and alpha-2 (COL2) chains. During post-transcriptional changes, proline and lysine

residues, as well as glucose and galactose, can be hydroxylated, and glucose and galactose

can be linked to hydroxylysine residues of collagen chains (Negari, Keshari, & Huang,

2021). The triple helix twists from the carboxyl end toward the amino end as a result of

temporary molecular chaperone attachment to procollagen chain domains. After secretion,

procollagen amino and carboxy peptides are broken down by proteinases, which eliminate

non-collagenous peptides. As collagen molecules are treated, electron microscopy can reveal

a banding pattern. The development of crosslinks between collagen trimers caused by post-
translational changes and the binding of other matrix components stabilizes fibril formation

(Veit et al., 2006).

Type II Collagen

Marine collagen and chicken collagen are both types II collagen. Researchers are still

researching the effectiveness of Type II collagen, but preliminary results have shown that

taking Type II collagen and acetaminophen together significantly reduces knee osteoarthritis

pain. It is also less tightly packed than Type I collagen, potentially indicating that the body

will be able to digest and absorb the collagen more efficiently in this form (Xu et al., 2021).

Despite Type II collagen's potential benefits for pain reduction and joint health, further

research is necessary regarding benefits including regulating joint inflammation, repairing

damaged cartilage, and increasing range of motion. The case of individuals with allergies to

fish or chicken, should consult their doctor before taking collagen supplements containing

Type II collagen (Negari et al., 2021).

Type III Collagen

Besides type I collagen, type III collagen is also a natural component of the body. Alpha

chains are unique to type III collagen.  All the others have multi-alpha chains. Besides Type I

collagen, Type III collagen is thought to be important for supporting the gut, muscles, and

blood vessels of the body. Bovine products comprise the largest amount of Type III collagen

(Omar, Malfait, & Van Agtmael, 2021).

The body will use amino acids in any way it needs to fight inflammatory diseases, so the role

supplements can play here is in doubt. Some studies show Type III collagen may be utilized
by the body to combat inflammatory diseases. It is not imperative to take collagen

supplements specific to certain areas of the body (Di Martino et al., 2022).

Type V Collagen

In the cornea, type V collagen plays a crucial role in controlling the size of collagen fibrils, so

that light can pass through smoothly. It is responsible for the formation and quality of fibers

in the body, as it works in conjunction with collagen Types I and III. The type V collagen

found in bones, corneal stroma, muscles, liver, lungs, and placenta is known to support the

matrix of bones, muscles, and placentas. There are positive results from research on Type V

collagen supplements, such as improved eye health, healthy skin, and healthy placental

tissues (Schiavinato, Przyklenk, Kobbe, Paulsson, & Wagener, 2021).

Even though scientists understand the way Type V collagen is used by the body, more

research is needed to determine whether Type V collagen supplements can be broken down

by the body for use in supporting these areas (Oh et al., 2021).

Type X Collagen

In the joint cartilage, type X collagen plays a role in bone formation. These networks can

indicate serious health conditions. Rheumatological disorders affecting bone and cartilage are

particularly prevalent in people with elevated levels of Type X collagen (Chen et al., 2021).

The type X collagen marketed by collagen supplement companies can be used to treat limb

injuries and broken bones. There's no hard evidence for this, as studies have only proved that

Type X collagen naturally found in the body contributes to bone formation and could be

useful in identifying rheumatoid diseases. Supplementing with Type X collagen does not

directly contribute to the healing of an injured area (Working et al., 2022).

Structure of collagen I:

Collagenous structures in the ECM include fibrillar, network, and transmembrane collagen

structures. The focus of our discussion is on fibrils made mostly of type I collagen.

At body temperature, monomers of type I collagen TC are unstable and adhere to random

coils. Collagen fibrillogenesis is thought to be the reason for the stability of triple helices.

Collagen's capacity to withstand stress in two, three, and four dimensions is also dependent

on the formation of strong macromolecular structures (Civil et al., 2021; da Silva Filipini,

Romani, Guimarães Martins, & Science, 2021). The essential rules for the production of

some forms of current collagen fibrils were established at least 500 Mya ago, according to

An, Kaplan, and Brodsky (2014), it emphasizing the importance of collagen fibrillogenesis.

The assembly of microfibrils, the intermediate size of collagen fibrils, results in the creation

of collagen fibrils in situ. Two concerns must be considered while studying the molecular

structure of collagen fibrils (Civil et al., 2021).

Collagen fibrils made mostly of type I collagen (all fibrous tissue except cartilage) have a

somewhat different structure than collagen fibrils made mostly of type II collagen (cartilage).

By studying solely type I collagen fibrils, we were able to identify the structure of thin

cartilage fibrils to intermediate resolution (4 nm) using recent data. The structure of cartilage

collagen fibrils shows a homotypic microfibril structure, with ten evenly spaced microfibrils

on each fibril surface and four equally spaced microfibrils in each fibril core (Hamanaka &

Mutlu, 2021).

Type I collagen tendon fibers are 500 nm in diameter and 106 nm in length. Type I collagen

triple helices have a diameter of 2 nanometers and a length of 300 nanometers.

Fibrillogenesis on a massive scale is required to produce collagen fibrils of natural size.

Fibrillar collagen is distinguished by its D-periodic structure, which has D = 67 nm. Because

TC monomers are not an exact multiple of D, but L = 4.46D, transmission electron

microscopy (TEM) images of collagen fibrils seem banded. This leads in 0.54D gaps and

0.46D overlaps. In structural models of collagen fibrils and microfibrils, these regular gaps

and overlaps must be taken into consideration (Salvatore et al., 2021).

It was first proposed as a three-dimensional structure to simulate collagen microfibrils.

Within a microfibril, this model is a two-dimensional stack with neighboring strands offset by

67 nm from each other. As demonstrated by TEM and atomic force microscopy, this model

accounts for gaps and overlaps within mature collagen fibrils (AFM). The three-dimensional

structure of type I collagen fibrils have been studied by several different research groups. A

number of fiber diffraction models, as well as TEM and AFM images of fibrils, have been

proposed (Xu et al., 2021).

Although the researchers agreed on the use of hexagonal unit cells with five TC monomers as

the foundation for a realistic model of the collagen fibril, they disagreed on key details. The

dispute over fibril structure may be coming to an end, according to new findings (Pedchenko

et al., 2021). Shoulders and Raines (2009)used synchrotron radiation to create a novel

electron density map of a type I collagen fiber, employing molecular anisotropic resolution.

Their findings show that collagen microfibrils contain quasi-hexagonal unit cells. The

supertwisted, right-handed microfibrils created by the molecular packing of TC monomers

result in the mature collagen fiber's spiral-like structure. As they point out, this model reveals

collagen fibrils to be a networked nanoscale rope (Shoulders & Raines, 2009).

Orgel and colleagues (2016) mapped the positions of collagen telopeptides in the N- and C-

terminal domains of TC monomers, then discovered that after being exposed to lysyl oxidase,
nearby telopeptides from the same TC monomer interacted and covalently cross-linked.

There might be cross-linking between and within microfibers. It's worth noting that the

nonhelical telopeptide portions preserve the collagen microfibril's super twisted character

(Shoulders & Raines, 2009; Terzi et al., 2018).

Previous attempts to extract collagen microfibrils from tissue samples failed due to

interdigitation and cross-linking of the collagen microfibers, making separation in an intact

form challenging. The new model also demonstrates that TC is present in collagen fibrils,

which are substantially more resistant to matrix metalloproteinase 1 (MMP1)-mediated

collagen proteolysis; the collagen fibril shields the collagen from MMP1. To get access to the

cleavage site of a TC monomer, MMP1 must proteolyze the C-terminal telopeptide of TC in a

fibril (Terzi et al., 2018).

Recombinant collagen production:

Collagens have been expressed using a variety of recombinant techniques. Due to their huge

size, multimeric nature, and modular design, collagen expression has proven viable in most

known expression methods, despite early doubts. Recombinant collagens are frequently

confined to biochemical characterization in basic research due to their manufacturing yield,

which has not yet been tailored to tissue engineering demands. Collagen heterotrimers can be

difficult to assemble in heterologous expression systems. Collagen homotrimeric molecules

are recombinantly synthesized in numerous systems, while heterotrimeric collagen molecules

are more difficult to create (Avila Rodríguez, Rodríguez Barroso, & Sánchez, 2018).

Recombinant proteins may be expressed in a variety of species, ranging from bacteria to

mammalian cells. Many posttranslational changes are required to achieve a suitably folded

multimeric conformation, which is critical for sustaining their activities and makes
recombinant collagen manufacturing difficult. It is therefore critical to select a cell expression

method to get a recombinant molecule that functions similarly to the natural protein.

Collagen's key structural property, as previously mentioned, is the hydrophobicity of the Gly-

X-Y triplet residues at the Y-position, which dictates its thermo-stability. The enzyme is

incapable of being used by prokaryotes and lower eukaryotes. Prokaryotic expression

systems, unlike other cellular approaches, are inexpensive and simple to scale (Tytgat et al.,

2020).

A variety of proteins are produced by yeasts such as Pichia pastoris and Saccharomyces

cerevisiae. Because yeast cells exude into the growth media, they are preferred over bacteria.

The stability of gene expression is assured by encoding the recombinant gene in the yeast

cell's genome. After a stable transformation of Pichia pastoris with enzyme subunits, fibrillar

collagens I, II, and III have been effectively expressed under heat conditions (Xi et al., 2018).

Using rod-shaped baculoviruses that can only infect specific insects, recombinant proteins

may be made rapidly and safely. Furthermore, proteins synthesized by baculovirus fold

appropriately, are disulfide-bonded and are distributed in the right subcellular compartments.

Despite their greater eukaryote status, insect cells lack prolyl-4-hydroxylase. When prolyl 4-

hydroxylase subunits are co-expressed with collagen chains I, II, and III in insect cells,

thermostable collagen I, II, and III trimmers develop (Ramshaw, Werkmeister, & Glattauer,

2019).

Both natural and synthetic collagen can be produced using a variety of expression systems.

Ramshaw et al. (2019) compiled information to compile a table outlining the production

levels achieved by each system. Expression systems that produce collagen-like polymers

instead of natural triple-helical collagens can produce production levels that are several

orders of magnitude higher. Yeast or bacterial expression systems are the clear choices for
maximum production when the triple-helical structure is not required. The incapacity of E.

coli to change post-translationally eukaryotic proteins, the potential for inclusion of bodies

(solid protein aggregates), and the likelihood of improper folding have all been noted as

drawbacks of E. coli expression methods (Pozzolini et al., 2015). However, because the

intended product (CLP3.1) is a single-stranded polypeptide to be soluble, post-translational

modifications, folding, and inclusion bodies are unlikely to be important. Ruggiero, et al.

used natural collagen pieces as well as a boost in E. coli expression as part of their E. coli

expression systems. The heparin-binding domain of the fragment was generated at a yield of

0.05 mg per 1 mL of bacterial cultures (with glycine per three amino acids). 1 Other glycine-

deficient fragments produced up to 0.5 mg per 1 mL of culture. GlytRNA levels in bacteria

may be too low to promote considerable quantities of collagen expression, according to one

theory (Wang, Lew, Premkumar, Poh, & Naing, 2017).

Xi et al. (2018) on the other hand, discovered that collagen carrying could be produced in E.

coli production systems in their investigation. The low levels of output found in E. coli

systems up to that point in time, according to Xi et al. (2018) were due to a paucity of studies

concentrating on improving the expression system.

The P4 gelatin was made highly hydrophilic by reducing the number of lysine residues, and

the pH of its gel matched the isoelectric point of genuine cow bone gelatin. P4 gelatin

contains a 36.8 kDa protein. Werten, et al. obtained recombinant gelatin expression levels of

up to 14.8 g/L using recombinant gelatin based on mouse type I and rat type III collagen

sequences. Gelatin yielded 11.3 g/L from a 5 copy transformant and 14.8 g/L from a 15 copy

transformant (Rutschmann et al., 2014). Proteases are capable of degrading the collagen

generated by the yeast expression system (both extracellular and intracellular proteases).

Ramshaw et al. (2019) were able to inhibit some deterioration in the high copy number
system by conducting fermentations at a relatively low pH (3.0 instead of 5.0). According to

Werten et al., the gelatin became less vulnerable to proteolytic cleavage after a site-directed

modification of the arginine residues in their P4 system (for example, the replacement of

Arg–Gly–Pro–Arg for Met–Gly–Pro–Arg resulted in the removal of cleavage products (An et

al., 2014).

In a different investigation, Banda et al. (2014) found that introducing PHI and NEU subunits

from human collagen to Bacillus brevis resulted in 0.5 g/L syntheses of collagen-related

proteins. In systems with short, repetitive sequences, the stability of recombinant genes with

lengthy, repeating sequences might be affected. E. coli is considered better than Bacillus

brevis in expressing these genes.

The amino acid makeup and coding sequence of a protein can have a big impact on the

production amount that can be produced in E. coli. It also has a substantial impact on protein

expression levels. E. coli systems will suffer if you need a high number of unusual proteins,

especially if they're coded for by rare codons. Natural proteins having highly repetitive

sequences, such as structural proteins, are likely to include rare codons. Martin et al.

developed an E. coli expression system to synthesize tropoelastin as a consequence (STEL).

To make the human tropoelastin sequence compatible with E. coli, a codon adjustment model

of the sequence was applied. Martin and colleagues were able to achieve expression levels of

20-30% as a consequence (Chan et al., 2022).

Ferrari, et al. created a long polypeptide with repeated sequences via the patent system. M.

Wade, H. Fausther-Bovendo, M.-A. De La Vega, and G. Kobinger (2021) then produced

polymers based on collagen-like polymers with a low proline concentration (45%) to combat

the problem of recurrent gene instability. E. coli plasmid vectors for encoding these genes

were created using tissue culture. The pJHL vector for thermally-inducible expression of
collagen was created using a plasmid reported in Cappello and Ferrari's patent as a starting

point. Yun et al. also created the plasmids pJY-1 and pJY-2 to express in the presence of

IPTG (Chan et al., 2022).  

Targeting viral vectors has undergone a paradigm shift, with a greater emphasis on systemic

targeting, which is more widely applicable and has enormous therapeutic promise. If a vector

is applied carefully, it must overcome several challenges to reach its target cells. The final

phase of infecting the target cell, which has received the greatest attention, is our emphasis.

For binding to the receptor on a target cell, an appropriate ligand must be incorporated into

the vector. Some viruses, such as the herpes virus for delivering neuronal gene copies, have

inherent tropisms that match their vector usefulness, but in many cases, the vector must be

modified to have a novel tropism (Tan et al., 2021).

Several vectors have made substantial progress in vector development, including lentiviruses,

adeno-associated viruses (AAVs), and adenoviruses. Due to comparable technological

challenges, strategies for improving the efficiency of one vector system are frequently

applied to improve the effectiveness of other viral vector systems. The primary techniques of

vector targeting are discussed here, as well as the significant hurdles that must be solved

before viral vector gene therapy may be applied in clinical settings (M. Wade, H. Fausther-

Bovendo, M.-A. De La Vega, & G. J. S. r. Kobinger, 2021).

HEK293 Cell line

Cell lines derived from human embryonic kidneys (HEK) have been immortalized by adding

snippets of adenovirus type 5 (Ad5) to human embryonic kidney tissue, creating HEK293.

There is evidence that 17% of the Ad5 genome has been integrated into the transformed HEK

cells' chromosome 19. Since its inception, this cell line has given rise to a number of
derivation cell lines with distinct properties. For protein transformation, numerous cell lines

are employed, including HEK293T and its variants HEK293E and HEK293F, as well as

HEK293S and HEK293H (Krzysztynska‐Kuleta, Olchawa, Sarna, & photobiology, 2021).

Previously, the HEK293 cell line was largely utilized to produce temporary and small-scale

amounts of research-grade proteins for scientific and pre-clinical studies. This cell line

possesses a variety of beneficial characteristics, making it a potential biopharmaceutical

manufacturer. It can create vast amounts of protein at a big scale, is repeatable across batches,

has fast reproduction, can adapt to a range of transfection techniques, and is, most

significantly, of human origin. The European Medicines Agency and the Food and Drug

Administration had approved five therapeutic proteins produced by HEK293 cells for human

use (Akbarian, 2022; Celik, Cora, Yigin, & Research, 2018).

Due to the inclusion of adenoviral DNA in its genome, this cell line has been proven to be

useful in the creation of viral vectors. Adenoviruses, influenza viruses, and lentiviruses have

all been created using this method. This cell line is an excellent host for membrane protein

production as well as a transient transfection tool for evaluating the pharmacological

properties of various receptor subtypes (Adham, Abdelfatah, Naqishbandi, Mahmoud, &

Efferth, 2021). It's an adherent epithelial cell line. Both adherent and suspension platforms

are now employed for expressing recombinant proteins since they have been adapted for

growth in suspension culture. Roller bottles, multilayered culture systems (e.g., Corning

CellStacks, Nunc Cell Factories, Greiner BIO-ONE and CELLdiscTM) and fixed bed

bioreactors are some of the attachment platforms that are accessible. Batch, fed-batch, and

continuous bioreactor operations are all examples of suspension platforms (Abaandou, Quan,

& Shiloach, 2021).

In mammalian expression systems, posttranslational modifications and heterologous protein

release in the culture medium are both feasible. For large-scale matrix protein synthesis,

HEK-293 cells are the most often employed cells. As we have shown for the first time for

collagen V homotrimer and other homotrimeric collagens, recombinant collagens made in

HEK-293 cells are frequently totally hydroxylated (Wang et al., 2017). HEK-293 cells have

already been shown to manufacture recombinant collagen V heterotrimer. However,

cotransfecting the pro1(V) and pro2(V) chains resulted in relatively modest amounts of

heterotrimeric collagen V while 15–20 g/ml homotrimer collagen V was formed in these cells

(Davison-Kotler, Marshall, & García-Gareta, 2019).

There is an adherent nature to this epithelial cell line. Recombinant proteins can be expressed

in both adherent and suspension platforms, which are optimized for growth in suspension

culture. In addition to roller bottles, multilayered culture systems (e.g., Nunc Cell Factories,

Corning CellStacks, Greiner BIO-ONE CELLdiscTM), and fixed bed bioreactors,

attachments can also be used. Among the suspension processes used in bioreactors are fed-

batch, batch, and continuous (Celik et al., 2018).

Interchain disulfide bonds develop between collagen's C-propeptides to generate molecular

filaments. Despite the presence of the C-propeptide in the recombinant pro2(V) chain, in the

absence of a reducing agent, the module did not move as a trimmer. The digested products

migrate to the location of the N- and C-propeptides after digestion with collagenase. Pro2(V)

chains, unlike pro2(I) chains, cannot form trimmers (Corcoran, Jordan, Carraher, Newcomb,

& biology, 2014).

Posttranslational modifications and heterologous protein release in the culture medium are

both possible in mammalian expression systems. HEK-293 cells are used most frequently for

large-scale matrix protein production. Collagens recombinant generated in HEK-293 cells are
often completely hydroxylated, as we have demonstrated for the first time for collagen V

homotrimer and other homotrimeric collagens (Wang et al., 2017). Recombinant collagen V

heterotrimer can already be produced by HEK-293 cells. When 15–20 g/ml homotrimer

collagen V was generated in these cells, however, cotransfecting the pro1(V) and pro2(V)

chains resulted in relatively minor quantities of heterotrimeric collagen V (Davison-Kotler et

al., 2019).

Because it is easy to transfect and may be converted to serum-free suspension culture, the

Human Embryonic Kidney 293T (HEK-293T) cell line is often used for the manufacture of

recombinant proteins and lentiviral vectors. An antigen mutant producing the SV40 large T

antigen was created in a HEK-293 cell line. One of the most important qualities of these cell

lines is their ability to proliferate fast and transfect successfully. Possessing the SV40 antigen

is also beneficial since it facilitates vector manufacturing. In fetal bovine serum-containing

culture conditions, HEK-293T cells are frequently cultured as static monolayers (FBS)

(Dalton & Barton, 2014). Adapting suspensions without serum is a feasible alternative for

overcoming these limits, as it allows for increased cell density without increasing surface

area, allowing for scale-ups. Several studies have shown that serum-free HEK-293T cell

culture promotes biopharmaceutical product development. Co-authors successfully produced

high-yield rFVIII using the HEK293SF-3F6 cell line. Using HEK-293 suspension adapted

cells and a culture media free of animal-derived components, a promising technique for the

creation of virus-like particles (VLP) for novel vaccination methods was obtained. The

potential of HEK-293 cells in suspension and serum-free culture media to produce high-titers

of infective influenza viruses was proven in a second fascinating investigation (Büssow,

2015).
Researchers have described multiple C1q receptors that interact with C1q CLRs, which might

lead to the recombinant production of C1q CLRs. For example, examining the CLR

contributions to C1q interactions between calreticulin and complement receptor 1 would

benefit greatly from such a technique. Both CLRs and GR are involved in these interactions.

The appropriate folding and phasing of the three collagen chains are important for the

synthesis of recombinant C1q CLRs. C1q GRs are admittedly important for registering CLRs.

As a result, removing the GRs may leave the collagen chains unregulated. Because the C1q

CLR is heterotrimeric, we substituted the C1q GR with a non-collagenous domain 2 (nc2) of

type IX collagen. In reality, this heterotrimeric domain is made up of three distinct helices,

and it has recently been discovered to have a role in collagen chain signaling. In contrast to

C1q GRs, NCC2 domains have no known features other than their potential to be

heterotrimeric. As a result, a well folded recombinant headless C1q is extremely beneficial

(Murphy et al., 2014).

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