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Question: biochemistry 1 protein sdspage questions 35...

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Biochemistry 1 (Protein SDS/PAGE)

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An antibacterial protein was isolated from a shore crab species. Eur J. Biochem. 264, 350-357 (1999) FEBS 1999 Purification and characterization of a cysteine-rich 11.5-kDa antibacterial protein from the granular haemocytes of the shore crab, Carcinus maenas Juliet M. Relf1, June R. S. Chisholm1, Graham D. Kemp2 and Valerie J. Smith1 Environmental&Evolutionary Biology, Gatty Marine Laboratory Unive rsity of St And rews, Fife, Scotland: Biomedical Sciences, North Haugh, University of St Andrews. Fife. Scotland Extracts of the granular haemocytes of Cacis maenas were subjected to ion-exchange chromatography and reverse-phase (RP)-HPLC to investigate the presence of an antibacterial protein of11 kDa. This protein was isolated, characterized and subjected to partial amino acid sequence analysis. It was found by mass spectrometry to have a molecular mass of 11 534 Da, to be cationic and hydrophobic and active only against marine Gram- positive bacteria. In addition its activity is stable after heating to 100 ℃ and is retained at concentrations as low as 10 μ g·mL-. II has an unusual amino acid sequence, unlike any known antibacterial peptide described in the literature but bears a consensus disulphide domain signature, indicating that it might be a member of the four- disulphide core proteins. Partial cDNA sequence data has been obtained. Keywords: antibacterial protein; Carcinus; crustacean; haemocytes; innate immunity The first step in the purification of this protein utilizes cation exchange chromatography. The procedure is shown below. Briefly explain why cation exchange is a good choice for this protein and why a salt (NaCI) gradient solution is used to elute or release the protein from the column. Purification The dialysed acid cxtract of granular cells (2 mL). with a protein conten of 800-900 ug mL. was applied to an 8 x 1 em CM Sepharose cation-exchange column equilibrated with 50 mM sodium phosphate (pH 6.5). Unbound material was eluted by extensive washing with buffer at a flow rate of 1mL-min Bound material was eluted with a two-step gradient of 50 mM sodium phosphate (pH 6.5) comaining 0-1 M NaCl as follows: 0-50% NaCI over 30 min and 50-100% NaCl over 15 min. Fractions (1 mL) were collected and absorbances read at 280 nm

3. The figure below is from the article. Fractions containing protein absorb UV light at a wavelength of 280 nm. Knowing the protein of interest is described as a cationic, in which fractions would you expect to find the antibacterial activity? Briefly justify your answer NaCl concentration os 04 03 02 0.1 100 Protein UV absorbance 0 0102030 5)607080 Fractions Further purification utilized size-exclusion chromatography and the purified material was then run out on a high resolution SDS/PAGE gel as described below Freeze-dried material was resuspended in 100 μL of deionized water, tested for antibacterial activity and run out on high resolution SDS/PAGE mini gels using a 16% separating gel, 10% spacer gel and 4% stacking gel [17]. To remove trace contaminants, the active fractions were buffered to a final concentration of 50 mM ammonium acetate (pH 6.5) and further purified by size-exclusion using FPLC (Pharmacia). A Superdex S-75 column equilibrated with 50 mM ammoniunm acetate buffer (pH 6.5) was used and separation was carried out at a flow rate of 0.5 mL-minT. Fractions (1 mL) were collected, freeze-dried and resuspended in 50 pL of deionized water 14.4 kDa 6.5 kDa 11.5 kDa-- 4. If the protein of interest had come off the size exclusion column pure, but in a later fraction, how would the gel in figure 4 have been different? Brieflyi5 aDa aastr justify your answer Fig. 4 A silver stained high resolution SDS/PAGE gel of the pure ucin fram the granudr atls Cn maenas. This sample was prepared by FPLC size exclusion on Superdex S75 after cation exchange and RP HPLC Standards are Bio-Rad wide range markers and the gel is stained with a Bio- Rad silver stain kit 5. What two roles does the SDS play in SDS/PAGE electrophoresis?

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