The Preparation Of Broad-spectrum Ultraviolet Absorbent Based On Streptavidin

Ultraviolet comes from high-energy solar radiation and it is invisible.According to the wavelength,it can be divided into UVA,UVB and UVC.Moderate ultraviolet(UV)can promote the synthesis of vitamin D in vivo.Excess ultraviolet will cause skin discomfort even diseases.For UV protection and development of sunscreen are gradually widely concerned,the preparation of broad-spectrum and efficient UV blocking and absorbent is one of the focus in current research about sun protection.Streptavidin(refered to SA)is a kind of secretory protein from Streptomyces avidinii.The secondary structure of the monomer is composed of eight antiparallel beta-chains,which fold to formate antiparallel β-barrel of the tertiary structure.The protein showed strong affinity to biotin,each barrel subunits can be combined with a biotin molecule.Four identical streptavidin protein monomers can associate to form tetramer.In streptavidin peptides,the apportion of aromatic amino acids is as high as 10.08%,especially tryptophan,which has a higher extinction coefficient for ultraviolet ray at 280 nm,is about 4.20%.Besides,tryptophan can absorb the energy transferred by tyrosine and release fluorescence.If the ultraviolet whose fluorescence peak near 340 nm further absorbed by compounds(such as coumarins),a new kind of broad-spectrum ultraviolet absorbent absorbing wavelength covers from 260 nm to 350 nm can be created.In this study,core SA sequence with natural activity was cloned.An analogous biotin short peptide sequence was connected to suppress SA in combination with intracellular biotin of the host,so as to ensure the normal metabolism of the biotin in host.In consideration of the bonding distance between biotin and analogous biotin short peptide,we selected sequence LEVLFQGP,which contains eight amino acids and specific recognized by human rhinovirus 3C protease as a connector between SA and analogous biotin short peptide.Genomic DNA of avidin streptomyces used as template to obtain the SA fragment with length of 357 bp by PCR.Taking the fragment as template,on the second PCR for SA1,which contains an analogous biotin polypeptide sequence.By site-directed mutagenesis(G59K,Y68G,L94K,W105S,HI 12K)from SA1 to SA2,the solubility increases through contents of lysine.Two vectors:pHND and pET-24a were tested to induce the expression of SA2 by 0.5 mM IPTG and purified.The induction condition of SA2 with pET-24a vector was optimized.The result showed that under the condition of 16℃,16h,SA2 expresssd more.Comparing the purification rate and purity,we found that SA2 was insuitable for Ni-NTA column.And the purity in recombinant protein with YND-tag(Yeast3’,5’-Bisphosphate Nucleotidase),also known as HAL2,which contains two tags for purification(Besides YND,it also has His-tag)is higher with high expression in the meantime.Thus,through purification of two affinity chromatography columns:Ni-NTA affinity column and PAP(3’,5’-Bisphosphate adenosine)affinity column,we obtained the high purity target protein SA2.7-Methoxycoumarin-3-carboxylic acid N-Succinimidyl Ester(MCSE),which is a kind of coumarins,was screening to label SA2 refering to relevant literature.Fluorescence spectra manifested,MCSE-SA2 could absorb UV280 and release 405nm fluorescence.This Suggest that fluorescence resonance energy transfer(FRET)had occurred between tryptophans(inside of SA2)and MCSE.All above,labelled protein MCSE-SA2 absorbed UV280 and UV340 through tryptophan and MCSE.Then it released harmless fluorescence of about 405 nm.The safety of the labelled protein was preliminarily explained on purpose of application.Conclusion mentioned above laid a foundation for discussing developing sunscreen using SA.Labelled streptavidin belongs to bioengineering product.It not only overcome some shortcomings of traditional chemical and physical absorbent,but strengthen absorptivity for UVA(utraviolet wavelength from 320 to 400 nm).The preparation of broad-spectrum utraviolet absorbent based on SA not only provides an alternative sunscreens,but take new idea for developing of new sunscreen in future.