Probing the interaction between recombinant human myelin basic protein and caseins using surface plasmon resonance and diffusing wave spectroscopy.
An intrinsically unstructured human myelin basic protein (hMBP) was expressed in the milk of transgenic cows (TGmilk) and found exclusively associated with the casein micellar phase. The interaction between the recombinant protein and milk caseins was investigated using surface plasmon resonance (SPR). An anti-human myelin basic protein antibody was covalently immobilized to the surface of the sensor chip. Subsequently the interaction between the recombinant protein (captured by this antibody) and caseins was studied in comparison to that noted with its human counterpart. Results showed a calcium-mediated interaction between the recombinant protein and caseins. The order of magnitude of this interaction was in agreement with the number of phosphorylated residues carried by each type of casein (alpha(s)- > beta- > kappa-casein). This selective interaction was not noted between the human protein and milk caseins indicating that the recombinant protein was phosphorylated to a higher extent than the human protein. The obtained results indicated that the co-expression of the recombinant protein and caseins by the mammary gland along with the recombinant protein's ability to form calcium bridges played a key role in the association of the recombinant human myelin basic protein (rhMBP) with the casein micelles of milk. Despite this association between the recombinant protein and milk caseins, light scattering investigations using diffusing wave spectroscopy (DWS) showed no significant differences between the milks of the transgenic and the non-transgenic control cows, with respect to both the average micelle size and surface charges. This was attributed to the low expression levels of the recombinant protein in milk.
Al-Ghobashy
Medhat A
MA
Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand.
Cucheval
Aurelie
A
Williams
Martin A K
MA
Laible
Götz
G
Harding
David R K
DR
D000818:Animals; D030801:Animals, Genetically Modified / Q000378:metabolism; D002364:Caseins / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D006801:Humans; D007700:Kinetics; D008823:Micelles; D004676:Myelin Basic Protein; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D013057:Spectrum Analysis / Q000379:methods; D020349:Surface Plasmon Resonance; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D002364:Caseins; C499048:MBP protein, human; D008823:Micelles; D004676:Myelin Basic Protein; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors