Biomedical Engineering Reference
In-Depth Information
sequence [
11
,
14
-
18
,
22
,
25
-
27
,
33
-
37
,
46
-
48
,
56
,
60
,
66
]. Recombinant synthe-
sis is advantageous when one desires to synthesize a longer sequence. Typically,
using SPPS to produce sequences with greater than 40 amino acid residues is dif-
ficult and requires synthesis by parts where the separate molecule parts are synthe-
sized separately with additional reactions [
3
,
19
-
21
,
23
,
24
,
26
,
37
,
42
,
61
,
67
,
68
].
Additionally, one can synthesize large amounts of peptide easily with recombinant
methods, a main advantage with recombinant DNA for peptide production over
solid phase peptide synthesis. The DNA sequences required to prescribe specific
amino acids in a peptide chain are known and established for the natural amino
acids and are constantly being discovered and engineered for non-natural amino
acids. To begin making a peptide sequence, the desired DNA sequence is con-
structed with primers, reproduced to create thousands of copies, and introduced
to plasmid DNA, which transfers the designed DNA sequence to
E. coli
colonies.
The colonies take up the plasmid DNA and begin producing the desired peptide
sequences. Numerous research groups using recombinant DNA peptide synthe-
sis have investigated the many possible different molecular products that produce
hydrogel materials after assembly [
32
,
45
,
48
,
68
,
69
].
The other method of peptide synthesis is commonly known as solid phase
peptide synthesis (SPPS). This method is synthetic and allows for more straight-
forward incorporation of noncanonical amino acids than with recombinant DNA
[
20
,
23
,
26
,
29
,
37
,
43
,
70
-
73
]. Additionally, the synthesis process can be done
by automation through machine or by hand. The convenience of using a machine
is the automated process of many repeat steps for longer peptide sequences. The
ease of use allows for a greater number of novel peptide sequences to be created.
Synthesis begins with a resin support made of small, porous polymer beads that
are functionalized so that the amino acid attaches with an amide bond. The func-
tionalization of the bead depends on the protection group used for the attaching
amino acids and cleavage conditions [
74
]. Amino acids are added sequentially
onto the solid phase-supported growing sequence, c-terminus to n-terminus. To
ensure only one coupling reaction per amino acid, the n-terminus of each amino
acid added to the reaction vessel has one of two possible protection groups, flu-
orenylmethyloxycarbonyl chloride (Fmoc) or tert-butyloxycarbonyl (Boc).
Traditionally, Boc was the protection group, but the need for hydrogen fluo-
ride (HF) in the final cleavage step brought about the Fmoc group for protection
[
74
,
75
]. The system is washed after each coupling step to get rid of unattached
amino acids, and then the n-terminus of the growing peptide chain is depro-
tected for the next amino acid. After the desired sequence is created, the resin and
attached amino acids go through filtration to get rid of unwanted amino acids with
the peptide sequence ultimately cleaved from the resin. As mentioned before, HF,
an acid, is needed in the cleavage step for Boc protected synthesis, while a base
such as piperidine is needed for Fmoc protected synthesis. Because of the sequen-
tial definition of amino acids by either synthesis method, peptide hydrogels can be
readily designed with different characteristics by specifically altering the primary
sequence. This primary structure design can affect directly the solution conditions
in which the peptides intermolecularly assemble, the nanostructure formed by the
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