Da-Hyeong Cho Protein Engineering Laboratory Department of Biotechnology and Bioengineering Sungkyunkwan University Site-Directed Mutagenesis

01. GFP & GFP Derivatives 03. Site-Directed Mutagenesis 02. pET Expression System Contents

Green Fluorescent Protein (GFP) laser.html Green fluorescent protein (GFP) traditionally refers to the protein first isolated from the jellyfish Aequorea victoria.

Wild-Type GFP GFP is a protein which exhibits bright green fluorescence when exposed to blue light. The protein composed of 238 amino acids (26.9kDa). The protein sequence and its resultant three-dimensional folding structure into the 11-strand β-barrel, is most likely crucial to the formation of the chromophore and its bioluminscene.

Wild-Type GFP The GFP from A. victoria has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm. Its emission peak is at 509 nm which is in the lower green portion of the visible spectrum.

GFP has the ability to exhibit intrinsic fluorescence thanks to three amino acids that cyclise (Ser65-Tyr66-Gly67) and then undergo an oxidation step during a complex maturation process. GFP Chromophore

Wild-type GFP (wtGFP) can be excited by light at two different absorption peaks, which the optimal excitation one is by UV light. This is problematic if one wants to use GFP in live cells, since UV radiation can damage them. Wild-type GFP folds poorly at 37°C, so it limits its usefulness for in vivo experiments in the lab. It can take up to four hours to fold GFP correctly Limitations of wtGFP

GFP Derivatives Changes in the amino acids in the fluorophore region, as well as in amino acids downstream from the fluorophore, result in changes in the color the fluorophore emits.

GFP Derivatives

Protein Expression

Genetic Elements Expression cassette  Promoter  Regulator binding site  Ribosome binding site (RBS)  Multiple cloning site (MCS)  Transcription terminator Antibiotic resistance gene Origin of replication Other control gene

pET Expression System The pET System is the most powerful system yet developed for the cloning and expression of recombinant proteins in E. coli. Target genes are cloned in pET plasmids under control of strong bacteriophage T7 transcription and (optionally) translation signals; expression is induced by providing a source of T7 RNA polymerase in the host cell. T7 RNA polymerase is so selective and active that, when fully induced, almost all of the cell’s resources are converted to target gene expression; the desired product can comprise more than 50% of the total cell protein a few hours after induction.

pET Expression System Promoter & Expression genes  lac promoter (host) – T7 RNA polymerase  T7 promoter (vector) – Target gene Regulator binding site (lac O) Control gene (lac I) T7 RNA polymerase inhibitor (T7 lysozyme)

pET Expression System

Mutagenesis Mutagenesis in the laboratory is an important technique whereby DNA mutations are deliberately engineered to produce mutant genes, proteins, strains of bacteria, or other genetically-modified organisms. Various constituents of a gene, such as its control elements and its gene product, may be mutated so that the functioning of a gene or protein can be examined in detail. The mutation may also produce mutant proteins with interesting properties, or enhanced or novel functions that may be of commercial use. Mutants strains may also be produced that have practical application or allow the molecular basis of particular cell function to be investigated.

Mutagenesis Methods of mutagenesis Random Targeted DNA recombination Point mutations Random change in residues or exhaustive enumeration of amino acid substitutions within selected sites

-> Random mutagenesis -> Point mutations in all areas within DNA of interest Result -> library of wild-type and mutated DNA (random) a real library -> many variants -> screening If methods efficient -> mostly mutated DNA Random Mutagenesis Screen mutants

Directed Mutagenesis -> Directed mutagenesis -> Point mutations in particular known area Result -> library of wild-type and mutated DNA (site-specific) not really a library -> just 2 species Point mutations - Substitution: change of one nucleotide (i.e. A-> C) - Insertion: gaining one additional nucleotide - Deletion: loss of one nucleotide

Polymerase Chain Reaction (PCR) An in vitro method for the enzymatic synthesis of specific DNA sequences Requires  Two specific oligonucleotide primers  DNA polymerase  dNTPs  Template DNA

Site-Directed Mutagenesis PCR Substitution Deletion Terminal Substitution PCR-based methods Site-directed mutagenesis by traditional PCR. Primers incorporating the desired base changes are used in PCR. As the primers are extended, the mutation is created in the resulting amplicon.

Site-Directed Mutagenesis

Primer design guidelines The mutagenic oligonucleotide primers for use in this protocol must be designed individually according to the desired mutation. The following considerations should be made for designing mutagenic primers  Both of the mutagenic primers must contain the desired mutation and anneal to the same sequence on opposite strands of the plasmid.  Primers should be between 25 and 45 bases in length, with a melting temperature (Tm) of ≥78 ℃.  The desired mutation (deletion or insertion) should be in the middle of the primers with ~10-15 base of correct sequence on both sides.  The primers optically should have a minimum GC content of 40 % and should terminate in one or more C or G bases Site-Directed Mutagenesis

Primer design example 5’-AGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTG-3’ 3’-TCTTCTTGAAAAGTGACCTCAACAGGGTTAAGAACAACTTAATCTACCACTACAATTACCCGTGTTTAAAAGACAGTCACCTCTCCCAC-5’ DNA sequence : AT->GC 5’-AGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTG-3’ 3’-TCTTCTTGAAAAGTGACCTCAACAGGGTTAAGAACAACTTAATCTACCACTACAATTACCCGTGTTTAAAAGACAGTCACCTCTCCCAC-5’ GC 5' GTCCCAATTCTTGTTGA TAGATGGTGATGTTAATG 3' 3' CAGGGTTAAGAACAACT ATCTACCACTACAATTAC 5‘ CG GC content: 40.54% Melting temp: 75.1 °C Mismatched bases: 2 Length: 37 bp Mutation: Substitution 5' flanking region: 17 bp 3' flanking region: 18 bp Site-Directed Mutagenesis

Experimental Scheme PCR Treatment of Dpn I Transformation into E. coli DH5a Plasmid preparation Transformation into E. coli BL21 (De3) Expression of EYFP EGFP → EYFP Products are checked by electrophoresis Making calcium competent cells (E. coli DH5a, E. coli BL21 (De3)) , , , 30

Materials & Methods 1.EGFP 와 EYFP nucleotide, protein sequence 찾아서 비교하기 번 amino acid 를 치환하기 위한 Primer design (GC content, Tm, base pair…) -> primer 표기는 5’ 부터 3.PCR method 정리 Report