110C Lec11 Recombinant Proteins & Protein Analysis Methods

110C Lec11 Recombinant Proteins & Protein Analysis...

Info iconThis preview shows pages 1–6. Sign up to view the full content.

View Full Document Right Arrow Icon
BCH 110C LECTURE 11 Recombinant Proteins Lodish 6 th ed. Chapters 3.6-3.7; 5.2-5.3; 5.5
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Recombinant protein expression and purification Clone a specific cDNA (i.e., the protein coding sequence of a gene without the introns) into a prokaryotic or eukaryotic “expression vector”. Introduce the expression vector into bacteria or eukaryotic cells Grow large numbers of cells that express the protein encoded by the cDNA Make cell extracts and purify the recombinant protein Use the recombinant protein for research or medicine (e.g., insulin, growth hormone)
Background image of page 2
Protein expression vectors e.g., Prokaryotic vectors Protein expression vectors have: Inducible promoters (+IPTG) Tags for affinity purification Histidines (Ni/Co metal binding) Epitopes (peptides recognized by antibodies) Proteins (e.g. GST) Coding sequence (cDNA) inserted in frame cDNA Amp r Ori PT5 : T5 promoter, lac O : lac operator, RBS : ribosome binding site, ATG : start codon, 6xHis/8xHis : His tag sequence, MCS : multiple cloning site, Stop Codons : stop codons in all three reading frames,
Background image of page 3

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Imidazole Interaction of Ni-NTA (nitrilotriacetic acid) matrix with histidine residues This is a specialized type of “ligand affinity chromatography”
Background image of page 4
Purification of His-tagged proteins expressed in E. coli E. coli cells lysed.
Background image of page 5

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Image of page 6
This is the end of the preview. Sign up to access the rest of the document.

This document was uploaded on 05/26/2011.

Page1 / 19

110C Lec11 Recombinant Proteins & Protein Analysis...

This preview shows document pages 1 - 6. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online