Construction of high level prokaryotic expression and purification system of PD-L1 extracellular domain by using Escherichia coli host cell machinery

Kalim, Muhammad; Chen, Jie; Wang, Shenghao; Lin, Caiyao; Ullah, Saif; Liang, Keying; Ding, Qi; Shuqing, Chen; Zhan, Jun

doi:10.1016/j.imlet.2017.06.004

Cited by 18 publications

(13 citation statements)

References 50 publications

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“…On other hands, larger NPs (>100 nm) are usually cleared from the circulation by phagocytosis. Similarly, very small NPs (<10 nm) have a high rate of clearance 39. The particle size of GA-NLC and both c(RGD) functionalized GA-NLC were found within the acceptable range, thus facilitating potentially passive targeting.…”

Section: Discussionmentioning

confidence: 94%

<p>Dimeric c(RGD) peptide conjugated nanostructured lipid carriers for efficient delivery of Gambogic acid to breast cancer</p>

Kebebe¹,

Wu²,

Zhang³

et al. 2019

IJN

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Background and purpose: Gambogic acid (GA) is a natural compound that exhibited a promising multi-target antitumor activity against several types of cancer. However, the clinical application of this drug is limited due to its poor solubility and low tumor cell-specific delivery. In this study, the monomeric and dimeric Cyclo (Arg-Gly-Asp) c(RGD) tumor targeting peptides (c(RGDfK) and E-[c(RGDfK) 2 ]) were used to modify GA loaded nanostructured lipid carriers (NLC) to reduce the limitations associated with GA and improve its antitumor activity. Methods: GA-NLC was prepared by emulsification and solvent evaporation methods and the surface of the NLC was conjugated with the c(RGD) peptides via an amide bond. The formulations were characterized for particle size, morphology and zeta potential, encapsulation efficiency and drug loading. The in-vitro cytotoxicity and cell uptake studies were conducted using 4T1 cell. Furthermore, the in-vivo antitumor activity and bio-distribution study were performed on female BALB/c nude mice. Results: The c(RGD) peptides modified GA-NLC was successfully prepared with the particles size about 20 nm. The HPLC analysis, FT-IR and 1 H-NMR spectra confirmed the successful conjugation of the peptides with the NLC. The in-vitro cytotoxicity study on 4T1 cells revealed that c(RGD) peptides modified GA-NLCs showed significantly higher cytotoxicity at 0.25 and 0.5 µg/mL as compared to unmodified GA-NLC. Furthermore, the cell uptake study demonstrated that better accumulation of E-[c(RGDfK) 2 ] peptides modified NLC in 4T1 cell after 12 h incubation. Moreover, the in-vivo study showed that c(RGD)s functionalized GA-NLC exhibited better accumulation in tumor tissue and tumor growth inhibition. In contrast to the monomeric c(RGD) peptide, the dimeric c(RGD) peptide (E-[c(RGDfK) 2 ]) conjugated GA-NLC showed the improved antitumor activity and tumor targeting ability of GA-NLC. Conclusion: These data provide further support for the potential clinical applications of E-[c(RGDfK) 2 ]-GA-NLC in breast cancer therapy.

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Section: Discussionmentioning

confidence: 94%

<p>Dimeric c(RGD) peptide conjugated nanostructured lipid carriers for efficient delivery of Gambogic acid to breast cancer</p>

Kebebe¹,

Wu²,

Zhang³

et al. 2019

IJN

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“…These interferons play a critical role in T‐cell proliferation and activation but the limited effects were observed with less consequences on tumor response either provoked later time. Expression and cloning of PD‐L1 also demonstrated that its expression likely involved in tumor evasion and might be responsible for contrasting clinical results (Kalim et al, 2017). Innate and adaptive immune responses play an efficient role in PD‐L1 expression as shown in Figure 3.…”

Section: Pd‐l1 Expression and Regulation In Cancermentioning

confidence: 99%

Programmed cell death ligand‐1: A dynamic immune checkpoint in cancer therapy

2020

Self Cite

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Antibody-based immunotherapies play a pivotal role in cancer research with efficient achievements in tumor suppression. Tumor survival is assisted by modulation of immune checkpoints to create imbalances between immune cells and cancer cell's environment. The modulation results in T-cell signal inhibition ultimately inert its proliferation and activation against various tumor cells. PD-L1, a 40 kDa transmembrane protein of B7 family, binds with PD-1 on the membrane of T cells which results in inhibition of T-cell proliferation and activation. PD-L1/PD-1 pathway has generated novel target sites for antibodies that can block PD-L1/PD-1 interactions.The blockage results in T-cell proliferation and tumor cell suppression. The PD-L1 immune checkpoint strategies' development, expression and regulations, signal inhibitions, and developmental stages of PD-L1/PD-1 antibodies are briefly discussed here in this review. All this information will provide a base for new therapeutic development against PD-L1 and PD-1 immune checkpoint interactions and will make available promising treatment options. K E Y W O R D Santibody, immune checkpoint, immunotherapy, PD-1, PD-L1

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“…Furthermore, prokaryotic expression system has been very mature with many advantages like as: simple operation, high yield, short production cycle, mature expression vectors easily available, and simpler purification methods (Rosano and Ceccarelli 2014). Among the prokaryotic systems, the pET system developed by Novagen Company is the most successful and products by this system have been in the market, which has produced great economic value (Hou et al 2006;Kalim et al 2017). In this study, the expression of iLRP was analyzed in detail by using this system, and the expression of three different vectors was compared.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of three different vectors on human iLRP expression in Escherichia coli

Liu

Dong

Yan

et al. 2020

Preprint

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iLRP (immature laminin receptor protein) is a tumor associated antigen over-expressed on the surface of most human cancer cells and plays an important role in the process of tumorigenesis and development. It has strong auto-immunogenicity in patients and is a good target protein for tumor immunotherapy. To find a practical and an efficient method for the production of recombinant iLRP, three expression vectors based on pET-30a(+) from pET expression systems were constructed. The first one is to add a 6xHis-Tag to the N-terminal of natural iLRP, which is called as pET-His-iLRP. The second one is to include an S-Tag between the C-terminal of 6xHis-Tag and the N-terminal of natural iLRP, which is called as pET-His-S-iLRP. The third one only contains the 6xHis-Tag in front of the N-terminal of iLRP, but the natural iLRP gene was first optimized according to the bacterial genome, and the constructed vector was named as pET-His-Opt-iLRP. Then the expression of three vectors in Escherichia coli (E. coli) BL21 (DE3) was analyzed. Results demonstrated that the iLRP expression was the highest in pET-His-Opt-iLRP, followed by pET-His-S-iLRP, and pET-His-iLRP expressed the lowest iLRP. This implied that the vector combining the codon-optimized iLRP gene with a 6xHis-Tag had the best effect on the expression of iLRP, and the expression in vectors with the natural iLRP gene depends on its leader sequences in the expression frame. It can be consequently concluded that the heterologous expression of human iLRP in a bacterial host could be greatly increased by genetically modifying gene constituents and using proper protein-tags, which laying the foundation on the future researches on its applications.

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Construction of high level prokaryotic expression and purification system of PD-L1 extracellular domain by using Escherichia coli host cell machinery

Cited by 18 publications

References 50 publications

<p>Dimeric c(RGD) peptide conjugated nanostructured lipid carriers for efficient delivery of Gambogic acid to breast cancer</p>

<p>Dimeric c(RGD) peptide conjugated nanostructured lipid carriers for efficient delivery of Gambogic acid to breast cancer</p>

Programmed cell death ligand‐1: A dynamic immune checkpoint in cancer therapy

Evaluation of three different vectors on human iLRP expression in Escherichia coli

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