Recombinant Pseudoalteromonas phage PM2 Protein P8 (VIII)

Basic Research Questions

• What is the genomic organization of Pseudoalteromonas phage PM2 and where does protein P8 (VIII) fit in?

  Bacteriophage PM2 has a genome consisting of three operons with specific regulatory elements. Transcription studies have shown that PM2 has early and late transcription events regulated by specific viral proteins. The PM2 genome has been mapped through cloning experiments involving overlapping DNA fragments in expression vectors. For structural proteins including P8, coding sequences have been successfully amplified by PCR and inserted into appropriate expression vectors for further study .

  Protein P8 (VIII) is one of several structural proteins encoded in the PM2 genome. While the search results don't specifically detail P8's exact genomic position, it belongs to a group of proteins (P3-P8) that have been studied through cloning and expression experiments .

• What methods are recommended for expressing recombinant PM2 Protein P8 (VIII)?

  Based on experimental protocols described in the literature, recombinant PM2 protein expression can be achieved through:

  Expression System	Vector Types	Induction Method	Temperature
  E. coli HMS174(DE3)	pRM expression vectors	IPTG induction	37°C
  E. coli DH5α with pDMI	pRM vectors	Induction specific to system	37°C

  For P8 specifically, the coding sequence should be amplified by PCR and inserted into appropriate expression vectors as demonstrated for PM2 proteins P3-P8 . The resulting constructs can be transformed into suitable E. coli strains like HMS174(DE3) for efficient protein production.

• What purification strategies are effective for recombinant PM2 P8 protein?

  While the literature doesn't specifically outline P8 purification, effective recombinant protein purification approaches can be adapted from similar systems:

  • Affinity chromatography using histidine tags (6xHis-tag) is recommended as a primary purification step

  • Tag removal can be performed using TEV protease with 1mM DTT

  • Final purification through size exclusion chromatography using columns such as HiLoad 26/60 Superdex 200 pg equilibrated with 20 mM HEPES (pH 7.5)/150 mM NaCl

  For confirming protein purity, SDS-PAGE analysis followed by Western blotting can be used to identify the target protein, similar to the approach used for other PM2 proteins .

• What culture conditions are optimal for Pseudoalteromonas phage PM2 propagation?

  For studying the native protein context, phage PM2 and Pseudoalteromonas cells should be cultured under the following conditions:

  • SB broth or marine defined rich medium

  • Temperature of 28°C

  • Strict aeration (critical for phage adsorption)

  • Media supplementation with 10 mM CaCl₂ to maintain phage viability when needed

  When studying viral adsorption specifically, it's essential to maintain vigorous aeration in normal growth medium resembling seawater. Without proper aeration, no virus binding will be detected .

Advanced Research Questions

• How can the structural characteristics of PM2 P8 protein be determined?

  Several complementary approaches can be employed to elucidate the structural features of PM2 P8:

  1. Computational prediction: Using methods like AlphaFold2 for initial structure prediction, providing insights into potential functional domains

  2. Experimental structure determination:

     • X-ray crystallography requiring high-purity protein samples

     • NMR spectroscopy for dynamic structural information

     • Cryo-EM particularly useful for studying P8 in the context of the intact virion

  3. Protein interaction studies:

     • Co-immunoprecipitation with other viral proteins

     • Cross-linking experiments followed by mass spectrometry

  When working with membrane-associated viral proteins like those in PM2, detergent screening may be necessary to maintain protein stability during structural studies .

• What functional assays can be used to characterize PM2 P8 protein's role in virus-host interactions?

  Several experimental approaches can be used to investigate P8's function:

  1. Adsorption assays: Measuring binding of labeled virions to host cells at different MOIs (multiplicity of infection), comparing wild-type and P8-modified virions. Adsorption rate constants can be calculated (e.g., 1.4×10⁻¹⁰ ml/min for ER72M2 cells and 2.2×10⁻¹⁰ ml/min for BAL-31)

  2. Transfection experiments: Following the protocol of van der Schans et al., PM2 DNA can be isolated and transferred into Pseudoalteromonas cells, with modifications to assess P8's role

  3. Radioactive labeling: Using L-[³⁵S]methionine (11 μCi/ml) added 10 minutes after infection or ³³P (11 μCi/ml) added 3 minutes prior to infection to track protein dynamics

  4. Zymogram analysis: For detecting potential lytic activities, particularly useful if P8 has enzymatic functions

• How does PM2 P8 protein contribute to viral membrane-host interactions?

  Investigating PM2's unique membrane structure requires specialized techniques:

  1. Electron microscopy: Thin-section electron microscopy of infected cells can visualize virus-cell interactions and membrane dynamics. No empty capsids are typically observed on cell surfaces during PM2 infection

  2. Sedimentation analysis: Using ³³P-labeled PM2 mixed with host cells (MOI 10) allows tracking of viral components, revealing that approximately 35% of radioactivity associates with cells while 65% is released

  3. Lipid-protein interaction studies: Since PM2 contains a membrane beneath its protein coat, techniques like liposome-binding assays could reveal if P8 interacts with specific lipids

  4. Mutational analysis: Creating specific P8 mutants to identify regions essential for membrane interactions

• What methods are effective for studying PM2 P8 protein's transcriptional context?

  To understand P8's expression and regulation:

  1. Primer extension analysis: Using labeled oligonucleotides to identify transcription start sites from viral RNA extracted at different time points post-infection. Protocol details:

     • Hybridize 15-30 μg total RNA with labeled oligonucleotide (1 pmol)

     • Mix in 40 mM PIPES (pH 6.8)-1 mM EDTA (pH 8.0)-0.4 M NaCl-80% formamide

     • Heat to 65°C for 5 min and cool slowly to hybridization temperature

     • Precipitate nucleic acids and resuspend in RT buffer with 5 mM MgCl₂ and 1 mM dNTPs

     • Add 30 U avian myeloblastosis virus reverse transcriptase

     • Incubate at 37°C for 30 min

     • Digest RNA with RNase A and analyze extension products on sequencing gels

  2. Transcriptional analysis: Identifying if P8 belongs to early or late transcriptional units, which would inform its functional role in the viral life cycle

  3. Promoter mapping: Determining regulatory elements controlling P8 expression

• How can site-directed mutagenesis be used to investigate PM2 P8 protein function?

  A systematic mutagenesis approach can reveal functional domains:

  1. Mutation design strategy:

     • Identify conserved residues through multiple sequence alignment

     • Target predicted structural elements (β-sheets, α-helices)

     • Focus on potential membrane-interaction domains

  2. Functional assessment:

     • Express mutant proteins in compatible E. coli systems

     • Test host cell binding with purified mutant proteins

     • Evaluate membrane association capabilities

     • Assess impact on virus assembly in complementation assays

  3. Experimental protocol considerations:

     • Design primers with appropriate restriction sites

     • Clone into expression vectors like those used for other PM2 proteins

     • Express in HMS174(DE3) or similar strains

     • Purify using affinity chromatography

• What bioengineering applications might utilize PM2 P8 protein?

  Recent research on P8 proteins from other sources suggests potential applications:

  Application Area	Approach	Key Parameters
  Cell proliferation	Culture medium supplementation	Concentration-dependent effects
  Wound healing	Activation of MAPK signaling	Enhanced cell migration
  Stem cell culture	Promotion of stemness markers	OCT4 and SOX2 expression

  While these findings relate to a different P8 protein (from Lactobacillus rhamnosus), they suggest potential research directions for investigating PM2 P8 protein's biomolecular interactions and possible biotechnology applications .

This FAQ collection provides methodological guidance for researchers working with recombinant Pseudoalteromonas phage PM2 Protein P8, from basic expression and purification to advanced structural and functional characterization techniques.