Chapter 6 Manipulation of Gene Expression in Prokaryotes

Molecular Biological Features for the Manipulation of Gene Expression Promoter and Terminator Ribosome binding site Copy number and Genetic stability Final location of the foreign protein Translational efficiency in the host organism Stability of the cloned gene protein

Host Cell E. coli Bacillus subtilis Yeast Animal cell Plant cell most well-known in its genetics, molecular biology, biochemistry, and physiology Bacillus subtilis Yeast Animal cell Plant cell Insect cell

Gene Expression from Strong and Regulatable Promoters Strong promoter RNA polymerase recognition and/or binding is strong. Transcription frequency is high.  more mRNA The sequence of the -35 and -10 regions are important for the strength of promoter.

Gene Expression from Strong and Regulatable Promoters Constitutive expression of foreign protein Can be detrimental to the host because of the energy drain Can cause plasmid instability Regulatable promoters control the rate of transcription E. coli lac, trp, tac (-10 lac + -35 trp) promoter Bacteriophage l pL promoter Bacteriophage T7 gene10 promoter

lac promoter Induced by lactose or IPTG (isoproply-b-D-thiogalactopyranoside) Repressed by glucose (catabolic repression) Glucose (↓)  cAMP (↑) cAMP (↑)  CAP-Promoter affinity (↑) CAP (catabolite activator protein) enhances the binding of RNA polymerase. lacUV5 a variant of lac promoter, stronger than wild-type lac promoter

Lac Promoter

trp promoter Derepressed by removing tryptophan or by adding 3-indoleacrylic acid (IAA) trp repressor Binding to the operator in the presence of tryptophan Leaky expression

tac promoter -10 region of lac promoter and -35 region of trp promoter Induced by lactose or IPTG Strength: tac > trp > lac (10: 3.3: 1)

Bacteriophage l PL Promoter Induced by shifting temperature from ~30oC to ~42oC Repressor : cI cI857: temperature-sensitive cI Active at low temperature (28~30oC) Inactive at high temperature (42oC)

Bacteriophage l PL Promoter

T7 gene 10 promoter (pT7) from bacteriophage T7 gene 10 Induced by IPTG pET vectors

T7 gene 10 promoter T7 promoter T7 RNA polymerase gene Transcription by T7 RNA polymerase T7 RNA polymerase gene In the E. coli chromosome on a bacteriophage l lysogen Under the control of the lac promoter Induced by IPTG

Increasing Protein Production * pKN402 * pPLc2833 ~ effective ori ~ strong promoter (pL) and high copy number multiple cloning site (MCS) (28oC: 82  42oC: 521) (fragment 1) (fragment c) ~ selectable marker (Ampr) (fragment 3) ↓↓ pCP3 (fragments 1, 3, and c)

pCP3

Large-Scale System Temp. shift Chemical inducer time & energy Chemical inducer high cost Inexpensive system is necessary for large scale protein production

Large-Scale System Two plasmid system (Figure 6.5) cI under the control of trp promoter Protein expression under the control of pL promoter molasses and casein hydrolysate (No tryptophan)  then, addition of trypton (containing tryptophan) Control gene expression Without Trp Repression of protein expression With Trp Induction of protein expression

Dual Plasmid System

Expression in Other Microorganisms The best promoter for use in a particular organism is not necessarily the one that is most efficient in E. coli (Table 6.2). Tn5 (70-bp DNA fragment from one of the terminal inverted repeats of transposon 5) functions in a wide range of bacterial hosts. Tn5 promoters can be used in a number of different bacterial host. Universal gram (-) bacterial expression vector Tn5 promoter in a broad-host-range plasmid pRK290 Efficient gene expression in different bacterial hosts