The Cloning, Expression, and Purification of ⍺-CA Nanobody for HIV-1 Capsid Assays Público

Abstract

In the face of escalating global infectious disease threats, there is a growing imperative to advance diagnostic told for early detection and treatment. Monoclonal antibodies (mABs), though pivotal for infectious disease detection and treatment, are hindered by their sizable nature, susceptibility to denaturation, and dependence on post-translational modifications, imposing constraints on their production and purification. Nanobodies, compact antibody fragments derived from Camelids, present a promising solution by overcoming the limitations of mABs. Their diminutive size and single-domain structure enable swift production in E. coli and an increased ability to detect biomarkers with exceptional sensitivity. The binding of the previously reported nanobody ‘59H10’ to the C-terminal domain of the HIV-1 Capsid protein (CA).12 To obtain an 𝛼-CA nanobody for HIV-1 capsid assays, an expression system was generated by cloning for the 59H10 nanobody. The coding sequence of the 59H10 nanobody was amplified using polymerase chain reaction and subsequently inserted into the pET24a vector by gibson assembly, yielding the 59H10 expression vector with diverse functional tags. Following transformation into NiCo21 (DE3) competent E. coli, the vector was sequenced to validate successful cloning. A construct of the nanobody with a 6x Histidine tag was purified from E. coli by a nickel column. The specificity and affinity of the nanobody was tested via biolayer interferometry. Our interest in 59H10 is due to the reported use of the 𝛼-CA nanobodies in stabilizing and solubilizing CASpyCat (or CASpyTag) modular capsid assembly system from Summers et al. (2019).13 The lab also has an interest in using this as a tool in future experiments for investigating HIV-1 capsid biochemistry.

Table of Contents

Table of Contents

Chapter 1: Introduction........................................................................................1

HIV-1 Structure and Mechanism of Entry...........................................................1

Figure 1.......................................................................................2

Figure 2.......................................................................................4

Figure 3.......................................................................................5

Figure 4.......................................................................................7

Nanobody vs. Monoclonal Antibodies (mABs)...................................................8

Figure 5 & 6.................................................................................10

Molecular Binding of 59H10.......................................................................11

Chapter 2: Materials and Methods..........................................................................14

Cloning of the 59H10 nanobody in pET24a......................................................14

Figure 7......................................................................................15

Figure 8......................................................................................16

Protein Expression and Purification of 59H10:6xHis..........................................20

HIV-1 CA121 Expression..........................................................................22

Biotinylating 59H10 nanobody for Bio-Layer Interferometry (BLI).........................23

Chapter 3: Results.............................................................................................25

Cloning of the 59H10 nanobody...................................................................25

Figure 9.......................................................................................26

Figure 10....................................................................................27

59H10:6xHis Expression and Purification.......................................................28

Figure 11.....................................................................................29

Figure 12....................................................................................30

Figure 13....................................................................................33

Figure 14....................................................................................34

Figure 15.....................................................................................35

BLI of 59H10 and HIV-CA121....................................................................36

Figure 16....................................................................................37

Figure 17....................................................................................38

Figure 18....................................................................................39

Chapter 4: Discussion........................................................................................41

Chapter 5: Figures............................................................................................43

Chapter 6: References........................................................................................46

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Molecular Biology, Virology
Palavra-chave	Antiretroviral Nanobody HIV-1
Committee Chair / Thesis Advisor	Stefan Sarafianos, Emory University
Committee Members	Edward Nam, Emory University Leila Rieder, Emory University

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