MRI is a powerful diagnostic tool that is non-invasive with deep tissue penetration. MRI
can provide detailed information about tissues and organs without utilizing any harmful
radiations. Due to low sensitivity, contrast agents are mostly used to enhance MRI images. In the
past decade, Dr. Jenny J. Yang’s lab has adopted and merged the idea of MRI contrast agents
with excellent knowledge of protein chemistry and developed novel class of Protein based MRI
contrast agents (ProCA). ProCAs can not only improve the low sensitivity with their dramatic
contrasting ability but also target the specific cancer biomarkers for early detection of cancerous
tissues using MRI. The first generation of ProCA was developed by constructing a Gd3+ binding
site on a stable scaffold protein, domain 1 of rat CD2. ProCA1 demonstrated 10 times higher
relaxivities as compared to the clinically used MRI contrast agents. High relaxivity, directly
corresponds to the use of low dosage, and low toxicity. Furthermore, targeted ProCA1 proved to
be excellent molecular imaging probe against breast cancer. During this process, a series of
ProCAs were developed with even more improved in vivo properties.
ProCA32 containing two metal binding sites is thermo-stable and has excellent solubility
and most importantly the production process is relatively easy. ProCA32 is known to accumulate
in the liver when injected into mice and dramatically enhances the liver region when observed
under MRI scanner. Thus, to further advance to the next stage of pre-clinical studies, rat based
ProCA32 must be humanized to reduce the immunological effects when injected into the human
body. The objective of this thesis is to optimize the production in the bacterial system and
examine functional and biophysical properties of hProCA32. There are several questions need to
be addressed for isolation of hProCA32 obtained from E.coli expression system. 1) What are the
optimal conditions for the cost effective and time efficient purification of hProCA32 and how to
isolate in its purest form under industrial set up?, 3) Does hProCA32 differ from rat ProCA32 in
terms of its structural properties, metal binding affinities, metal selectivity, and stability and
relaxation properties as a MRI contrast agent?, 4) How to reduce the possible in vivo
immunogenicity effects generated by our contrast agent? 5) Can modification of hProCA32 with
PEG improve it’s in vivo stability, solubility, bio-distribution and biocompatibility without
disrupting the functioning of hProCA32? 6) Can site- specific cysteine PEGylation be applied to
hProCA32 to attain homogeneity? 7) How different is hProCA32 from the cysteine PEGylated
hProCA32 and which length of PEG is best suitable for the in vivo application with altering its
metal-binding properties?
One of the major findings in this thesis is that hProCA32 and its variants can be
successfully expressed and purified using the bacterial system. The optimal expression can be
achieved using both BL21 (DE3) and BL21 (DE3).pLySs competent cells at reduced
temperatures after induction. In purification process, boiling the soluble hProCA32 supernatant
indeed showed the removal of major unwanted proteins. Furthermore, the protein was
successfully isolated from nucleic acids using 3 % streptomycin and affinity column with a
maximum final yield of about 32 mgL-1. In addition, hProCA32 can be isotopically labeled with
15
N using M9 minimal medium. Labeled hProCA32 with 15N can be used for lanthanide binding
studies using NMR as a future direction.
Another major finding is for metal binding studies, which suggests that hProCA32 has
high binding affinity for Gd3+ with dissociation constant of 10-22 M as compared to the other
physiological metals such as calcium (10-8 M) and zinc (10-6 M). The metal selectivity Log
(K
Gd/KCa) of hProCA32 for Gd 3+
is higher over calcium (Log (K
(K
Gd/KZn)= 15.34). Various fluorescence metal binding assays were tested to develop an indirect
metal binding assay. The relaxivity remained relatively comparable with rat ProCA32 with r1 of
27 mM-1s-1 and r2of 36 mM-1s-1. Cysteine PEGylated hProCA32 showed excellent serum
stability at 37 °C up to 12 days. However, these are preliminary results and must be repeated to
draw any concrete conclusions. Furthermore, 2 K cysteine PEGylation of hProCA32 showed T1
weighted enhancements and T2 weighted properties when introduced into mice under MRI
scanner. Another finding, suggests that hProCA32 can bind to trivalent Lu3+ with a dissociation
constant of 10-22 M, which proposes a possible application for our contrast agent in SPECT.
All these significant findings are supportive of hProCA32’s functional similarities with
rat ProCA32 and it is evident that hProCA32 can prove to be a great candidate for future
molecular targeting. The long-term goal is to optimize the stability by using suitable PEG length,
which does not disrupt the physical properties and enhances the relaxation properties of
hProCA32. Additionally, the binding ability of hProCA32 with other metals such as Gallium,
Yttrium, Zirconium and Indium can be tested as future direction for its application in nuclear
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