Structure-Function Studies of the ORF1 Protein From the Insertion-Site Specific Retroposon M5 Found in Indo-Pakistan Urban Malarial Vector Anopheles stephensi
Akinbosede, Daniel Oluwatosin
In the 1950s Barbara McClintock inferred the occurrence of transposition: the movement of small segments of DNA - entities known as transposable elements from one position of the genome to another (McClintock, 1950). Classification of transposable elements in regards to mechanism of transposition distinguishes them into two groups; transposons (Class II) and retroposons (Class I). The term retrotransposon was coined as it illustrates the transposition of these elements is dependent on the reverse transcription of RNA to DNA through a reverse transcriptase, also known as the ‘copy and paste’ transposition. The M5 retroposon has been found in numerous mosquito species such as Anopheles stephensi. M5 present in these Anopheles is a class 1, non-LTR transposable element of the jockey clade family with two open reading frames (ORF). Due to its APE like endonuclease, M5 should transpose to random sites of the genome. However, in A. stephensi the element has been reported to transpose with site specificity. The aim of the project was to gain structural and functional information on the role of the ORF1 protein of M5 in order to understand the element’s site specificity. To perform functional and structural studies, an Escherichia coli expression vector was designed with a synthetic AsM5 ORF1 insert. Heterologous expression and purification of ORF1p in E.coli produced signs degradation or very low yield of the unfolded protein possibly due to the host’s inability to process some eukaryotic features required for the protein. Saccharomyces cerevisiae was then chosen as an expression system for AsM5 ORF1p production. The AsM5 ORF1 gene was cloned from the E.coli expression vector into the pYES2/CT S.cerevisiae expression vector and sequencing verified that the AsM5 ORF1 insert was successfully cloned into the pYES2/CT vector. Optimization of the lysis and expression protocol in S.cerevisiae had slowed progress but a highly effective method of cell lysis was developed. Expression of the full length ORF1p in S.cerevisiae was not confirmed and difficulties in expression could be attributed to the fact that the original synthetic ORF1 sequence which was cloned is codon optimised for expression in E.coli hindering expression in S.cerevisiae. CPSF100_C was one of the conserved domains identified in the AsM5 ORF1 amino acid sequence using conserved domain web tools. For further analysis the CPSF100_C domain was cloned into an E.coli vector and successfully expressed in rich media, the protein was the purified using immobilized metal ion affinity chromatography (IMAC) and ion exchange chromatography (IEC). In order to progress to NMR studies of the domain,15N labelled expression of CPSF100_Cp was performed. Usually, several expression showed the non-peptide fusion partner glutathione S-transferase (GST) being expressed without the protein of interest possibly due to mRNA instability when the gene is expressed in minimal media. The identification of protein domains such as CPSF100_C and their interactions with nucleic acids and other proteins will likely be the key to understanding AsM5’s site specific retrotransposition.
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