Heterologous expression

1. Heterologous expression Heterologous expression means that a protein is expressed in a cell that does not normally make (express) that protein.

2. Heterologous expression systems • Escherichia coli • Yeast (Saccharomycescerevisiae, Pichiapastoris etc) • Dictyosteliumdiscoideum (have circular nuclear plasmids packaged in a nucleosomal structure) • Xenopusoocytes (South-African clawed toad) • Insect cells (infection of insect cells by recombinant baculoviruses) • Mammalian cell lines (COS cells, an engineered line of African green monkey kidney cells) COS stands for CV-1 origin, SV-40(virus)

3. Factors affecting expression of heterologous proteins • trans-acting factors and cis-acting elements • Affinity tag used • Size of the heterologous protein • Source of the heterologous protein • Codon biasing • E. coli host strain used • Culture conditions • Degradation of the heterologous protein

4. 1) trans-acting factors and cis-acting elements Cis-acting factors are intrinsic to the DNA strand, whereas trans-acting factors are produced elsewhere in the genome. Response elements are short sequences of DNA where specific transcription factors binds.

5. Cis-acting response elements - several genes transcribed coordinately (e.g.storage protein or photosynthetic protein family) have sequence modules in common that control the coordinate regulation. These modules are called response elements. These elements are a class of cis-acting elements. • Features of Response Elements: • contain short consensus sequences • modules are related but not identical • not fixed in location but usually within 200 bp upstream of the transcription start site • a single element is usually sufficient to confer a regulatory response • can be located in a promoter or an enhancer • assumed that a specific protein binds to the element and the presence of that protein is developmentally regulated 1a) trans-acting factors and cis-acting elements

6. Trans-acting factors- factors, usually considered to be proteins, that bind to the cis-acting sequences to control gene expression Role of trans-acting factors: factor may be expressed in a specific tissue manner (spatial regulation) factor may be expressed in at specific time in development (temporal regulation) factor may require modification (phosphorylation) factor may be activated by ligand binding Interact with environmental signal 1b) trans-acting factors and cis-acting elements

7. 2) Affinity tag used • Affinity tags permit a variety of proteins to be purified using easy procedures. • Small affinity tags (His-tag) do not interfere with protein expression. • Affinity tags of origin other than bacteria are less expressed.

8. 3) Size of the heterologous protein • Proteins less than 100KDa are well tolerated in E. coli and hence are expressed to significant levels in the bacterium. • Proteins more than 100KDa are unable to express properly in the bacterium and hence are degraded. • If the size is exceeding this limit then host systems other than E. coli should be used.

9. 4) Source of the heterologous protein • Many eukaryotic proteins are not appreciably expressed in E. coli. • Membrane proteins are also unable to express properly in E. coli. In order to express such proteins other expression systems (yeast, insect or mammalian cell lines) are used.

10. 6) Codon biasing • In a particular organism a specific codon is preferably used to code a specific amino acid, despite of the other codons. • Optimization of the codon can be done by in vitro gene synthesis thus removing the rare codons. • Site-directed-mutagenesis can be used if the number of rare codons in the gene is less.

11. 6) E. coli host strain used • Choosing the proper E. coli host strain is obligatory for the substantial expression of recombinant proteins. It should be protease deficient. • The most commonly used protease deficient strain is BL21. • Certain affinity tags require specific E. coli host strains. For example, TB1 E. coli host strain is used to express proteins tagged with maltose binding protein (MBP).

12. 7) Culture conditions • Optimizing the culture medium (like LB or 2YT) is mandatory for the expression of heterologous proteins in E. coli. • Proper aeration should be maintained. Optimum is 20% rule. • Volume of the culture media should not exceed the 20% of the maximum capacity of the flask. • Induction at proper growth phase also affects protein expression. Mostly, mid log phase cultures are used for induction. • Concentration of inducer should also be empirically determined.. • Proper temperature also affects the expression. Grow the cultures at 37ºC and after induction grow at 23-30ºC.

13. 8) Degradation of the heterologous protein The recombinant protein may not properly fold and hence is degraded by the bacterial proteases. To minimize proteolysis: • Use of protease deficient strain, • Growth of host strain at low temperature, • Co-expression of molecular chaperons, • Replacement of specific amino acid residues to eliminate protease cleavage sites, etc.