Research Experiences

Chapter 2 The Practise and Theory of Protein Crystallography.pdf

Research Experiences and Research Interests ( More Research Activities on CPD)

1. Research Experiences

Postdoctoral Research Fellow, The Role of Envelope N-glycosylation in HIV Transmission and Vaccine Development, The HIV-1 Envelope Structure-Function and Viral Fitness Research Group, from 2013 to 2015, Department of Molecular and Cell Biology, The University of Cape Town

Principle investigators: Dr. Zenda Woodman and Dr. Brandon Weber

HIV: Structure and Function and Viral Fitness Research Group

The Role of Envelope N-glycosylation in HIV Transmission and Vaccine Development

Alternative to codon optimization in heterologous protein expression systems

Recombinant Sub-Unit Vaccines: Monomeric and Trimeric HIV Envelope Vaccines

  • The RV144 HIV trials, commonly known as Thai HIV trials, provided promising results when 31 % of Phase III trial participants were protected from HIV infection

  • The current immunogenicity studies involving monomeric gp120 and trimeric gp140 demonstrates cross-clade NAb responses with substantially higher potent titers induced by gp140 trimers than gp120 monomers for a diverse set of both tier 1 and tier 2 viruses.

  • A functional gp41-gp120 interaction is observed in monomeric but not oligomeric, uncleaved HIV-1 Env gp140

  • Cleaved gp140 monomers exhibit function and internal structural order consistent with those expected on infectious HIV envelope glycoprotein. Thus uncleaved gp140 oligomers are not an accurate representation of the pre-fusion, soluble form of infectious HIV envelope glycoprotein.

  • Cleavage is required for Env function, and cleaved trimers interact more efficiently with broadly neutralizing antibodies (bNAbs) compared to uncleaved trimers and less efficiently with non-neutralizing antibodies.

  • Envelope glycoprotein ectodomains dictate many of the antigenic and structural features

  • Thus, cleaved monomeric or trimeric gp140 envelope glycoproteins are indispensable in structural studies and vaccine development

  • Due to HIV envelope glycoprotein structural and glycosylation profile differences between transmitter/founder and chronic viruses, vaccine development needs to focus on these HIV variants, "the original antigenic sin," which is currently not the case. This study will address this disparity in HIV structural studies and vaccine development.


Due to the paucity of CD4 T cells at the genital tract, HIV variants are successfully transmitted because they carry an optimal arrangement of High Mannose-type N-glycans that facilitate their uptake by dendritic cells through binding to DC-SIGN, transfer to the lymphoid tissue, and enable the trans-infection of CD4+ T cells. Furthermore, cleaved native-like trimers of gp140 from both transmitter/founder and chronic viruses will lead to cross-clade improved vaccines for HIV.


Cleaved native-like trimers of gp140 will lead to cross-clade-improved HIV vaccines.

Trimeric forms of HIV envelope glycoproteins are indispensable in structural and vaccine studies. HIV envelop glycoprotein trimers result from the processing of gp160 into gp120 and gp41 subunits, which forms noncovalent interactions on the surface of the HIV and are crucial to HIV transmission and infectivity. Due to the instability of the native gp140 trimers, the most common way to make them is to remove (mutate) the cleavage site between the gp120 and gp41 subunits. Current data show that doing so creates trimers that adopt irregular, nonnative configurations. The other commonly used method is to produce cleaved, stable timers linking gp120 with gp41 subunits using a disulfide bond. Trimers made in this way are yet to be assessed in great detail for their ability to elicit bNAbs in preclinical trials.

On the other hand, cleaved, stabilized trimers, without disulfide bond linkage between gp120 and gp41 (i.e., with noncovalent interactions between gp120 and gp41), in contrast, resemble the native spikes on the HIV-1 virus. Our findings will help structural and vaccine programs by showing how to make native-like trimers that can significantly impact HIV structural and vaccine studies. The rationale for vaccine trials based on monomeric gp120 and uncleaved gp140 trimers must be re-evaluated.


  • Expression and purification of native-like gp140 trimers of transmitter/founder (T/F) and chronic (C) viruses that will help in structural and vaccine programs

  • The role that envelope N-glycosylation plays in HIV transmission by comparing transmitter/founder and chronic viruses glycosylation profile and their role in binding to DC-SIGN

  • Biophysical and biochemical characterization of native-like gp140 trimers

  • Antigenic analysis of T/F and C native-like gp140 and monomeric gp120

  • Pseudovirus neutralization assays (paving the way for HIV preclinical and clinical vaccine trials)

Objective 1. Compare the type of carbohydrate structures at N-glycosylation sites of gp120 from transmitted founder variants and those from chronic infection

Rationale: DC-SIGN binds to gp120 high mannose N-glycans during transmission and not complex sugars, which is selected during chronic infection

Experimental Approach

1. Expression of envelope glycoproteins in mammalian cells

2. Purification of envelope glycoproteins

3. Analysis of N-glycosylation patterns by mass spectrometry

Objective 2. Identify the N-glycan sites essential for binding to DC-SIGN

Rationale: transmitted founder viruses carry an optimal arrangement of N-glycans that is lost at chronic infection

Experimental Approach

1. Site-directed Mutagenesis of N-glycan sites involved in binding to DC-SIGN

2. Investigate the binding of mutants to DC-SIGN

3. Expression, purification, and mass spec analysis of mutants as in Objective 1 to confirm the identity of N-glycans

Objective 3. Biophysical and biochemical differences between transmitter/founder and chronic HIV-1 subtype C gp140 and gp120 envelope proteins

  • The crystal structure of the trimeric native-like gp140 Env spike has not yet been determined. There is also very limited biochemical and biophysical data available on these trimers.

Objective 4. Antigenic analysis of purified native-like gp140, gp120, and pseudovirus neutralization assays (paving the way for preclinical and clinical HIV vaccine trials)

HIV-1 Subtype C Envelope Glycoproteins to be used in this study

1) Cap177 T/F (acute) vs. Chronic

2) Cap239 T/F (acute) vs. Chronic

3) Cap210 T/F (acute) vs. Chronic clones X2

Doctor of Philosophy in Molecular Biology and Biotechnology, 2009-2012, Department of Molecular Biology and Biotechnology, The University of Sheffield

Supervisor: Prof David P. Hornby

Advisors: Prof C. Neil Hunter FRS

Prof Stuart A. Wilson

  • Structural biology of the membrane transport proteins

  • Molecular biology of Enterococcus faecalis V583 proton-dependent manganese transporter (MntH) protein

  • Peptide mass fingerprinting

  • Mass spectroscopy ESI TOF MS

  • Preparation of over-expressed membrane proteins for thermodynamics studies

  • Physicochemical studies of MntH important for membrane proteins crystallography

  • Membrane proteins for 2-D crystallization

  • AAS and ICPAES

  • Techniques to improve membrane proteins crystallization

  • Membrane proteins expression, purification, physicochemical studies, and structural determination showed that protein stability and interactions are very important

Master of Philosophy in Molecular Biology and Biotechnology, 2008-2009, Department of Molecular Biology and Biotechnology, The University of Sheffield

Supervisor: Prof David P. Hornby

Advisors: Prof C. Neil Hunter FRS

Prof Stuart A. Wilson

  • Techniques in membrane proteins' structural determination

  • Bacterial membrane transport proteins

  • Expression of proton-dependent manganese/divalent cation transport protein

  • Solubilization and purification of membrane proteins

  • Membrane protein monodispersity analysis and N-terminal sequencing

  • Circular dichroism spectroscopy and substrate binding

  • Thermal stability studies (advantages and disadvantages of different thermal stability techniques)

  • Membrane proteins crystallization

Master of Science in Molecular and Cell Biology, Feb to Dec 2007, School of Molecular and Cell Biology, The University of Witwatersrand, Johannesburg

Supervisor: Prof Monde Ntwasa

  • Identification and isolation of dung beetles, Euoniticellus intermedius (Coleoptera: Scaeabaeidea) from cattle farms

  • Establishing dung beetle cultures and monitoring the developmental biology of embryos

  • Extraction and spectroscopic quantification of total RNA

  • Analysis of RNA (RNA gel electrophoresis)

  • cDNA synthesis and construction of a cDNA library

  • Cloning of the blunt-ended cDNA into pGEM-T Easy

  • Preparation of chemically competent cells

  • Transformation of E. coli XL1-Blue cells with recombinant pGEM-T Easy-cDNA Beetle Library

  • DNA purification and sequencing of cDNAs

  • Bioinformatics (analysis of cDNAs antimicrobial peptides open reading frames)

BSc with Honours in Biochemistry and Cell Biology, Feb to Dec 2006, School of Molecular and Cell Biology, The University of Witwatersrand, Johannesburg

Supervisor: Dr. Jonathan Burke

  • PCR to make copies of c-Jun N-terminal kinase 1 open reading frame (ORF) cloned in pGEX-4T-3-JNK1 and pcDNA3 α46 so that it can be subcloned in pQE-30 AU (The JNK1 ORF was cloned in frame with HA-tag in pcDNA3 α46 and GST-tag in pGEX-4T-3-JNK1 while pQE-30 UA had a 6xHis tag)

  • Plasmid analysis using restriction enzyme digestion and agarose gel electrophoresis

  • Transformation of E. coli bacteria cells and protein expression optimization

  • Protein (inclusion bodies) unfolding using chemical denaturants and protein refolding in different buffer conditions

  • Protein purification, quantification, and analysis using spectroscopic probes (absorption, fluorescence, and CD)

  • Studies of protein unfolding and refolding events using fluorescence spectroscopic techniques

  • Functional studies of refolded protein using enzyme kinetics

  • Bioinformatics (Protein and DNA analysis)

  • Understanding protein folding events can prevent the formation of inclusion bodies during heterologous recombinant protein expression

2. Research Interests

  • HIV Transmission and Vaccine Development

  • Biophysical and biochemical dynamics of biological membranes

  • Synthetic biology applicable to membrane proteins structure and function

  • Membrane proteins folding and insertion into the lipid bilayer

  • Mechanisms of membrane transport protein

  • Mechanisms of antimicrobial peptides