Development of therapeutic protein drugs is the new emerging field that has been
revolutionized in the past few decades[48]. The recombinant technology and protein
engineering have been used to alter and enhance the native properties of a desired protein and
utilize it for the disease related treatments. For instance, recombinant human insulin is used to
treat diabetes mellitus, recombinant erythropoietin is used to treat anemia due chronic renal
failure or chemotherapy and many more with special targeting and diagnostic properties. [49,
64] However, these proteins are not naturally evolved for therapeutic drug application,
modifying them leads to an immune response by potential antibodies formations. Such immune
response is referred to as immunogenicity. According to the guideline by FDA, there are several
patient-specific factors and product-specific factors that influence immunogenicity that
influence immunogenicity[65]. The patient-specific factors such as genetic background of the
patient such as patient’s immunologic status, competence, sensitivity, tolerance, route, dose and
frequency of the administration differ from patient to patient and are difficult to be studied. The
product specific factors include: the origin of the protein (human or non-human sequence),
glycosylation,and PEGylation. The product related factors could be optimized and monitored
while developing the protein drug.Humanization of a therapeutic protein drug is an important
method to reduce the immunogenicity. Among our previously established ProCAs, ProCA32
seemsto be the best candidate to be carried on to the next step of designing a therapeutic protein
drug, and test biophysical properties. In our approach of testing the humanized ProCA32,
PEGylation is another technique that is FDA-approved to reduce the immunogenicity effects.
As mentioned earlier in chapter1, when protein drug is conjugated with PEG, flexible and inert
PEG chains wraps up the antigenic epitopes of the drug molecule reducing the toxicity and
immunogenicity generated by foreign protein drugs and decreases the degradation caused by
proteolytic enzymes without interfering in its therapeutic properties.[50]PEG is a hydrophilic
molecule, which increases the solubility and increase in molecule weight help extend the half-
life of the protein that enhances the stability with reduced renal filtration. Our previously
PEGylated versions of ProCAs demonstrated an increase in the relaxivity due to increase in the
molecular weight, changes in water properties and water-metal exchange rate due to hydrophilic
PEG. Protein modification by PEG can be done on various residues, but the most frequently
employed approach is PEG conjugation with free amine groups typically on lysine. However,
the majority of proteins sequence consists of multiple lysine residues (hProCA32 consists of 16
lysine residues as seen in Figure 4.16) and PEG specific to amine can react with any free amine
available in the sequence. This leads to the formation of multiple isomers of PEGylated-proteins
with multiple amine bearing sites being modified. Separation of such heterogeneous mixture of
PEGylated protein becomes difficult. If the modification of these therapeutic proteins does not
attain reproducibility, the regulatory FDA approval becomes complicated. In order to overcome
by site-specific PEG conjugation at cysteine. This approach allows selective protein
modification at a single or predetermined site with the homogenous outcome. Cysteine residues
are mainly associated with disulfide bridges and formation of secondary structures; hence,
accessible cysteine residues without any function are rarely available. A protein sequence
containing free cysteine residue, which is not involved in the disulfide bond formation and does
not play an important role in the protein functioning and activity can be used for modification.
Moreover, a cysteine residue can be introduced in the protein sequence either replacing a non-
essential amino acids from the protein sequence or insertion at a desirable position can be easily
attained by site-directed mutagenesis. In case of hProCA32, a cysteine residue was inserted at
the N-terminus to allowsite-specific conjugation of a single PEG at thiol group on cysteine
using maleimide-PEG. Forward and reverse primers were designed, as shown in the Figure
4.17A; DNA gel electrophoresis results in (Figure 4.17 B) shows a band at
6000bpcorresponding to expected template DNA. This indicates that the molecular cloning
experiment was successful. The plasmid construct was further confirmed by getting it
commercially sequenced from GENWIZ. The DNA sequence obtained from the sequencing was
analyzed and aligned by Blast and Clustalw. The DNA and protein sequence alignment in
Figure 4.18 shows the desired mutation at C-terminal of hProCA32 was successfully inserted
Figure 4.16 Possible Lysine (Cyan) and Cysteine (Beige) PEGylationsites on hProCA32. Xray crystalized structure obtained from pdb file 1RK9 and modified using USCF Chimera S56D, D101E and F103W
Figure 4.17 Molecular cloning of hCA32.cys by PCR (A) Forward and Reverse Primer design for site directed mutagenesis of hCA32 (B) DNA gel electrophoresis of the plasmid obtained after PCR
Figure 4.18 DNA and protein sequence alignment of mutated variant hCA32.cys with hCA32