You have been given an unknown peptide by a colleague and asked to provide an amino acid sequence. Using the experimental data shown below, deduce the peptide sequence giving the proper order of the amino acids and including the position of any disulfide bonds. Using your best deduced sequence, provide the complete chemical structure for the unknown peptide (ie. before hydrolysis). You may not be able to provide the sequence for ALL amino acids. Indicate which amino acids for which you are uncertain. (NOTE: Amino acids given in peptide fragments are the result of the complete hydrolysis with HCl + HEAT and are given in alphabetical order, not in order of primary structure). Expt. 1: Chymotrypsin digestion of the unknown peptide resulted in 3 peptide fragments. The peptides were separated, treated with β-mercaptoethanol and hydrolyzed overnight in HCl. The following results were obtained: Frag. 1 (after hydrolysis): Arg, Asn, Glu, Leu, Thr Frag. 2 (after hydrolysis): Asp, Phe Frag. 3 (after hydrolysis): Cys, His, Lys, Met, Trp NOTE: β-Mercaptoethanol treatment of individual fragments above did not result in multiple fragments. Expt. 2: Trypsin digestion of the unknown peptide resulted in 3 fragments that were separated, treated with β-mercaptoethanol and hydrolysed overnight in HCl. The following results were obtained: Frag. 4 (after hydrolysis): Arg, Cys, Leu, Trp Frag. 5 (after hydrolysis): Asn, Glu, Thr Frag. 6 (after hydrolysis): Asp, His, Lys, Met, Phe NOTE: β-Mercaptoethanol treatment of individual fragments above did not result in multiple fragments. Expt. 3: Cyanogen Bromide treatment resulted in: Frag. 7 (after hydrolysis): Arg, Asn, Cys, Glu, His, Leu, Lys, Thr. Trp Frag. 8 (after hydrolysis): Asp, Met, Phe NOTE: β-Mercaptoethanol treatment of individual fragments above did not result in multiple fragments. Expt. 4: Carboxypeptidase released Glu
You have been given an unknown peptide by a colleague and asked to provide an amino acid sequence. Using the experimental data shown below, deduce the peptide sequence giving the proper order of the amino acids and including the position of any disulfide bonds. Using your best deduced sequence, provide the complete chemical structure for the unknown peptide (ie. before hydrolysis). You may not be able to provide the sequence for ALL amino acids. Indicate which amino acids for which you are uncertain. (NOTE: Amino acids given in peptide fragments are the result of the complete hydrolysis with HCl + HEAT and are given in alphabetical order, not in order of primary structure). Expt. 1: First cycle of Edman’s degradation on the unknown peptide results in Proline. Expt. 2: Trypsin digestion of the unknown peptide resulted in 2 fragments that were separated, treated with -mercaptoethanol and hydrolyzed overnight in HCl. The following results were obtained:
Frag. 1 (after hydrolysis): Arg, Cys, His, Ile, Lys, Met, Phe, Pro, Thr, Trp, Tyr Frag. 2 (after hydrolysis): Glu, Leu, Lys, Met, Trp NOTE: Treatment of Frag. 1 with -mercaptoethanol gave: Frag. 1a (after hydrolysis): Arg, Cys, Pro, Thr, Trp Frag. 1b (after hydrolysis): Cys, His, Ile, Lys, Met, Phe, Tyr Expt. 3: Chymotrypsin digestion of the unknown peptide resulted in 2 fragments that were separated, treated with -mercaptoethanol and hydrolysed overnight in HCl. The following results were obtained: Frag. 3 (after hydrolysis): Glu, Ile, Leu, Lys, Met, Trp Frag. 4 (after hydrolysis): Cys, His, Met, Pro, Trp, Tyr Frag. 5 (after hydrolysis): Lys Frag. 6 (after hydrolysis): Arg, Phe, Thr
NOTE: Treatment of Frag. 4 with -mercaptoethanol gave: Frag. 4a (after hydrolysis): Cys, Pro, Trp Frag. 4b (after hydrolysis): Cys, His, Met, Tyr Expt. 4: Cyanogen Bromide treatment resulted in: Frag. 7 (after hydrolysis): Arg, Cys, His, Met, Phe, Pro, Thr. Trp Frag. 8 (after hydrolysis): Ile, Leu, Lys, Met, Tyr Frag. 9 (after hydrolysis): Glu, Lys, Trp NOTE: -Mercaptoethanol treatment of individual fragments from Expt 4 did not result in multiple fragments.
Reagent Trypsin Chymotrypsin Cyanogen Bromide Carboxypeptidase
Cleavage Site C-side of Lys, Arg C-side of Phe, Tyr or Trp C-side of Met C-terminal amino acid
A schematic of the vector p7012 is shown. The restriction enzymes listed cut only where indicated; they do not cut anywhere else in the vector or insert.
b) In which of the strategies would Gene W be inserted into the vector in only one direction?
