GEN 314

1
GEN 314

• Gene Manipulation Lecture Four
• Gene expression in E. coli

2
Introduction

• Requirements for expression in E. coli
• transcription of cloned insert (gene must be
  placed under control of promoter recognized by
  hosts RNA polymerase 3-end terminator)
• Translation requires
• that an mRNA bears a ribosome binding site
  (rbs)
• mRNA includes translational start codon (AUG
  or GUG)
• sequence complementary to 3-end of 16S rRNA
• Concerns of expressing proteins in E. coli
• post-transcriptional modifications, e.g.
  phosphorylation (modifications can be introduced
  in E. coli)
• stability some proteins are degraded if
  their config. is doesnt protect from E. coli
  proteases
• rate of formation of functional 3D structures
  chaperones form correct tertiary structures (E.
  coli!!!)

3
Fusion proteins

• Clearly, for expression to occur
• structural gene placed under promoter
• promoter function in E. coli
• protein synthesis from N-terminus
• However, fusion proteins may also be produced
• two sequences that are in-frame relative to
  one another and expressed as a single protein
• Advantages of expressing fusion proteins
• N-terminal fusion may stabilize the protein
  from proteolytic degradation
• E. coli protein on the N-terminus can
  facilitate purification of the foreign protein
  activity (pET31b Zhao et al. 2007. Prot. Expr.
  Pur. 53 (1) 232-237)
• or act as a reporter gene to monitor
  expression (e.g. luciferase van Leeuwen et al.
  2000. Plant Mol. Biol. Rep. 18 143a-143t)
• in some cases, it may be possible to cleave
  fusion protein to yield native protein

4
Luciferase reporter system

• Luciferase are group of enzymes commonly used in
  nature for bioluminescence
• Firefly luciferase, from the firefly Photinus
  pyralis
• Light is produced by the oxidation of a pigment
  (luciferin), involving ATP
• reaction catalyzed by luciferase
• Two step reaction
• luciferin ATP ? luciferyl adenylate PPi
• luciferyl adenylate O2 ? oxyluciferin AMP
  Light
• Reaction very energy efficient because nearly all
  energy input gt light

5
Achieving gene expression

• Plasmid-based systems gene expression achieved
  via,
• cloning DNA cartridge containing the necessary
  regions for efficient transcription and
  translation
• cloning gene of interest in specially-designed
  expression vector
• Commonly use expression vectors, e.g. pET vectors
• promoters used include lac, trp, lpp, etc
• Tests to show that expression of a cloned gene is
  under control of selected E. coli promoter
• expression obtained only in correct
  orientation!
• expression should be enhanced by environmental
  conditions that normally lead to promoter
  activation, e.g. IPTG with lac-based expression
  vectors or starvation in trp-based vectors

6
pET expression vector
www.emdbiosciences.com
7
Reading frame

• Expression of fusion proteins depends largely on
  the correct translational reading frame
• For example, plasmid pMR100 (designed to
  facilitate the detection of inserts in the
  correct reading frame)
• lac promoter directs transcription of
  NH2-terminus of lacI gene followed by a MCS and
  an out-of-frame lacZ gene
• gene insertion into the MCS results in
  frameshift production of functional
  ?-galactosidase
• lacZ gene acts as a reporter of functional
  activity
• Other reporter systems include,
• alkaline phosphatase
• luciferase
• galactokinase
• chloramphenicol acetyl transferase

8
Fusion proteins in pBR322

• Clone cDNA transcripts into the PstI site (rat
  preproinsulin mRNA Villa-Komaroff et al. 1978)
• PstI lie with the ApR gene
• Cloning via homopolymer tailing
• Different lengths of repeating nucleotides (e.g.
  GC) added and some may be in the correct reading
  frame

9
Detecting gene expression

• Unknown gene (function) three methods
• mini-cells small, spherical, anucleate cells
  produced continuously during growth of some
  mutants strains of bacteria
• size-difference separation from wild-type cells
  by sucrose gradient
• mini-cells from plasmid-free parents lack DNA
• mini-cells from plasmid-containing parents are
  capable of RNA and protein synthesis ideal for
  detecting gene expression from recombinant
  plasmids
• UV irradiation of the host cell (maxi-cell
  method Sancar et al. 1979) most popular method
  
• Irradiate E coli recA uvrA cells gt stop DNA
  synthesis but plasmid not irradiated remain
  capable of normal DNA replication.
• Application infect irradiated bacteria with ?
  vector containing insert gt cloned genes seen
  against background of ?-specified proteins
• in vitro transcription and translation systems
• Known gene hybridization techniques (e.g.
  Western blot) sequence available for probe
  design

10
Western blot
Determine conc., boil (unfold proteins), add
buffer soln, sulfhydryl soln (prevent di-sulfide
bonds) SDS (detergent -ve charge around
proteins)
11
End Lecture Four