Native Protein Analysis on TIMSTOF Pro mass spectrometer v2

Method described in David Robert et al., https://pubs.acs.org/doi/10.1021/jacs.1c02713

Towards the Creation of a Transgenic Possum Parasite

<p>The brushtail possum. Trichosurus vulpecula, is New Zealand's most serious vertebrate pest; possums destroy native flora and fauna and are vectors of bovine Tb. Conventional control is considered to be unsustainable and, in the long term, biological control is seen as the only solution to reducing possum numbers. The aim of this project is to contribute to the development of a self-disseminating vector that will spread a control molecule throughout the possum population reducing fecundity or increasing mortality. The possum-specific parasite Parastrongyloides trichosuri has considerable potential a-s such a vector. A protein from P. trichosuri specifically, was found to be antigenic in possums. The antibodies to this protein were purified from positive possum serum and used to detect the antigen on the surface of infective larvae but not in the excretory/secretory products of either larvae or adults. The protein was isolated from crude infective larvae and found to show homology to the heat-shock 70 family of proteins. Genomic DNA was extracted, an oligonucleotide probe made and a genomic library screened for the Hsp70 gene. Several positive clones were found and DNA isolated and sequenced from one such clone. Five kilo bases of unambiguous sequence was obtained in which was an open reading frame of 2 kb. Theoretical translation of this gave a protein of 64 amino acids with 80% homology to the Hsp70A protein of C. elegans. The region upstream of the ATG initiator codon was amplified and 1.3 kb of the putative promoter region was cloned into a vector containing the gfp:lacZ reporter genes. This construct was microinjected, first into C. elegans to demonstrate promoter function, and then into both tree-living and parasitic adults of P. trichosuri. Reporter gene expression was shown in the progeny of microinjected parasitic adults. RNA was made from infective P. trichosuri larvae, reverse transcribed and the coding sequence for the PtHsp70 protein cloned into an expression vector and expressed in E. coli, The recombinant protein pattern had a similar pattern of trypsin digestion products as the native protein, as shown by MALDI-TOF mass spectrometry, but it was immunologically distinct from the native protein. The culmination of this project was the generation of a transgenic P trichosuri, the first vertebrate endoparasitic nematode to be heritably transformed. This is a necessary step in the development of a self-disseminating vector to be used in the biocontrol of possums.</p>

Towards the Creation of a Transgenic Possum Parasite

<p>The brushtail possum. Trichosurus vulpecula, is New Zealand's most serious vertebrate pest; possums destroy native flora and fauna and are vectors of bovine Tb. Conventional control is considered to be unsustainable and, in the long term, biological control is seen as the only solution to reducing possum numbers. The aim of this project is to contribute to the development of a self-disseminating vector that will spread a control molecule throughout the possum population reducing fecundity or increasing mortality. The possum-specific parasite Parastrongyloides trichosuri has considerable potential a-s such a vector. A protein from P. trichosuri specifically, was found to be antigenic in possums. The antibodies to this protein were purified from positive possum serum and used to detect the antigen on the surface of infective larvae but not in the excretory/secretory products of either larvae or adults. The protein was isolated from crude infective larvae and found to show homology to the heat-shock 70 family of proteins. Genomic DNA was extracted, an oligonucleotide probe made and a genomic library screened for the Hsp70 gene. Several positive clones were found and DNA isolated and sequenced from one such clone. Five kilo bases of unambiguous sequence was obtained in which was an open reading frame of 2 kb. Theoretical translation of this gave a protein of 64 amino acids with 80% homology to the Hsp70A protein of C. elegans. The region upstream of the ATG initiator codon was amplified and 1.3 kb of the putative promoter region was cloned into a vector containing the gfp:lacZ reporter genes. This construct was microinjected, first into C. elegans to demonstrate promoter function, and then into both tree-living and parasitic adults of P. trichosuri. Reporter gene expression was shown in the progeny of microinjected parasitic adults. RNA was made from infective P. trichosuri larvae, reverse transcribed and the coding sequence for the PtHsp70 protein cloned into an expression vector and expressed in E. coli, The recombinant protein pattern had a similar pattern of trypsin digestion products as the native protein, as shown by MALDI-TOF mass spectrometry, but it was immunologically distinct from the native protein. The culmination of this project was the generation of a transgenic P trichosuri, the first vertebrate endoparasitic nematode to be heritably transformed. This is a necessary step in the development of a self-disseminating vector to be used in the biocontrol of possums.</p>

Tetraphenylporphyrin Enters the Ring: First Example of a Complex Between Highly Bulky Porphyrins and a Protein

Tetraphenylporphyrin (TPP) is a synthetic porphyrin whose properties can be readily modified, endowing it with significant benefits over naturally occurring porphyrins. Yet, their insolubility in water and/or steric bulk have rendered them incompatible with biological systems. Herein, we report the first example of a native biomolecule capturing TPP as well as its derivatives. The haemoprotein HasA, secreted by certain pathogens to scavenge haem from their hosts, can capture various metal- and meso-substituted TPPs. The rapid crystallisation of TPP derivatives captured by HasA revealed the binding mode of TPP at excellent resolutions. A single-site mutation (L85A) of HasA enlarged the binding pocket, allowing the incorporation of a bulkier derivative of TPP. HasA binding TPP derivatives was also demonstrated to inhibit proliferation of the opportunistic pathogen Pseudomonas aeruginosa. This study not only represents a simple method for the complexation of TPP derivatives with a native protein, but also opens the door for the future use of TPP derivatives as biological tools.

Analyses of Cell Type-Specific Effects of MicroRNA-298 on Native Protein Expression Via Truncated 3’UTR Hold Translational Promise

Alzheimer’s disease (AD) is marked by neurofibrillary tangles and senile plaques comprising amyloid β (Aβ) peptides. However, specific contributions of different cell types to Aβ deposition remain unknown. Non-coding microRNA (miRNA) play important roles in AD by regulating major proteins involved, like Aβ precursor protein (APP) and β-site APP-cleaving enzyme (BACE1), two key proteins associated with Aβ biogenesis. MiRNAs typically silence protein expression via binding specific sites in 3’- untranslated region (3’UTR) mRNA. MiRNA regulates protein levels in a cell-type specific manner; however, mechanism of miRNA’s variable activities remains unknown. We developed “miRNA-associated native protein expression” (miRnape) assays to determine a natural "UTR limit" for a miRNA’s function in a particular cell type. We report that miR-298 treatment reduced native APP protein levels in an astrocytic but not in a neuronal cell line. From miR-298’s effects on APP-3’UTR activity and native protein levels, we infer that APP 3’-UTR length could explain the differential miR-298’s activity. Such truncated, but natural, 3’-UTR found in a specific cell type provides an opportunity to regulate native protein levels by particular miRNA. Thus, miRNA’s effect tailoring to a specific cell type bypassing another undesired cell type with a truncated 3’-UTR would potentially advance translational research.

Making Weak Antigens Strong: Preparing Immune Complexes for Injection

If antibodies against a particular antigen are available, that antigen can be purified and used for further immunizations, and antigens thus purified can show enhanced immunogenicity. Purified immune complexes can be injected directly, or while coupled to beads; the presence of antibodies and/or beads stimulates phagocytosis and usually will not influence the response. This method provides a useful means of antigen enrichment for a variety of applications, such as using antibodies raised against a denatured antigen to harvest a native protein for further immunizations, or when using a monoclonal antibody as an intermediate to the preparation of polyclonal antisera. Injecting antibody-coated antigens has also been used to mask a particularly immunodominant epitope on an antigen, and thereby develop a response against other epitopes. The amount of antigen needed to elicit a strong response using immune complexes will vary from one compound to another. Doses as low as 50 ng of antigen have been used successfully when delivered this way.

Synthesis of Epitope-Targeting Antibody Based on Native Protein–Protein Interactions for Ftsz Filamentation Suppressor

Phage display and biopanning is a powerful tool for generating binding molecules for a specific target. However, the selection process based on binding affinity provides no assurance for the antibody’s affinity to the target epitope. In this study, we propose a molecular-evolution approach guided by native protein–protein interactions to generate epitope-targeting antibodies. The binding-site sequence in a native protein was grafted into a complementarity-determining region (CDR) in the antibody, and a nonrelated CDR loop (in the grafted antibody) was randomized by phage display techniques. In this construction of antibodies by integrating graft and evolution technology (CAnIGET method), suitable grafting of the functional sequence weakly functionalized the antibody, and the molecular-evolution approach enhanced the binding function to inhibit the native protein–protein interactions. Antibody fragments with an affinity for filamenting temperature-sensitive mutant Z (FtsZ) were constructed and completely inhibited the polymerization of FtsZ. Consequently, the expression of these fragments drastically decreased the cell division rate. We demonstrate the potential of the CAnIGET method with the use of native protein–protein interactions for steady epitope-specific evolutionary engineering.