Molecular Biotechnology: Principles and Applications - Glick, B., Pasternak, J. 2002

Fundamentals of Molecular Biotechnology
Optimization of Gene Expression Cloned in Prokaryotic Systems
Conclusion

To obtain a specific protein product, it is necessary to ensure the proper METABOLISM/31.html">Transcription of its coding Gene and the Introduction/27.html">Translation of the corresponding mRNA. Initiating transcription at the correct site requires a promoter, whereas its termination requires a stop codon. A cloned gene often lacks such signal sequences, meaning both must be provided for its expression in a prokaryotic host Cell. Furthermore, because most biotechnological Applications require the protein to be produced in large quantities, a promoter must be used that yields a high level of transcription (a strong promoter) and is recognized by the host cell's RNA polymerase. Continuous Transcription of the cloned gene depletes the energy resources of the host cell; therefore, promoters whose activity can be regulated either by specific low-molecular-weight compounds or by Temperature changes must be employed.

The efficiency of Protein Synthesis depends on the presence of specific nucleotide sequences within its mRNA. To prevent the degradation of the protein product or to facilitate its secretion, cloned genes encoding the protein are subjected to targeted modifications. This may involve attaching a ribosome binding site upstream of the Transcription initiation site (which itself may also need to be added) or appending a termination codon to the end of the cloned gene to ensure translation arrest. If protein secretion is desired, a signal sequence must be inserted upstream of the cloned gene, ensuring its reading frame is in phase with that of the target gene.

Another challenge is the low stability of Proteins encoded by cloned genes. Recombinant Proteins can be cleaved by host cell proteinases. To prevent this, the cloned gene can be modified so that one or more additional Amino Acids are appended to the N-terminus of the protein molecule. In this form, the recombinant protein is no longer susceptible to such rapid degradation. In addition, these "extra" amino acids sometimes assist in the subsequent purification of the fusion protein, for example, via immunoaffinity Column Chromatography. The junction between the components is designed so that the molecule can be cleaved (chemically or enzymatically) at that site to yield the components in pure form.

Most microorganisms used to produce protein products grow only in the presence of oxygen. Oxygen is poorly soluble in Water and is rapidly depleted during rapid growth. To overcome this limitation, two approaches have been used: 1) employing strains incapable of synthesizing certain Proteolytic Enzymes, and 2) introducing genes encoding *Vitreoscilla* sp. Hemoglobin into the host cell genome, which binds ambient oxygen and increases its intracellular concentration.

As the copy number of a cloned gene increases, The amount of synthesized product also increases. However, when scaling up to large-scale production, the plasmid/cloned DNA construct is very frequently lost. To prevent this, Methods have been developed to integrate the cloned gene into the host Organism's chromosome. In this case, the gene remains within The Cell as an integral part of the host DNA.

The introduction and expression of foreign DNA in a host organism often disrupt its metabolism and normal functioning—a phenomenon known as metabolic load. A variety of methods have been developed to minimize this effect while simultaneously optimizing the yield of the target protein and the Stability of the transformed Cells.

Expression systems are highly diverse, and researchers must continuously optimize conditions tailored to producing a given protein in a specific host organism. Nevertheless, despite differences in details, the same core techniques are used to construct A wide variety of expression systems.

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CONTROL QUESTIONS

1. What methods can be used to influence the expression of genes cloned in prokaryotic organisms?

2. What is the lacI^q gene and how is it used?

3. Why is a plasmid vector with the strongest possible promoter not always the best expression vector?

4. What is the tac promoter and how is its regulation carried out?

5. The p^L promoter of phage λ, which naturally infects only E. coli, is nevertheless sometimes used as a component of broad-host-range expression vectors. How can the p^L promoter be "adapted" to initiate transcription in other organisms?

6. Sometimes the strategy for target protein synthesis involves producing it as part of a chimeric product. What are the advantages of this approach, and how are Chimeric Proteins engineered?

7. What are inclusion bodies, and how can their formation be prevented?

8. What is the advantage of displaying foreign proteins on the cell surface? What strategies are used to render proteins secretable?

9. How can multiple copies of a gene be inserted into a single plasmid?

10. How can The problem of oxygen supply be solved for E. coli cells synthesizing high levels of a foreign protein?

11. A target sequence can be integrated into chromosomal DNA in two ways: 1) on its own; 2) as part of a plasmid carrying this sequence. How does each of these events occur? What

advantages or disadvantages are associated with the integration of a plasmid vector into the host DNA?

12. What are metabolic burdens, and what causes them?

13. Propose several ways to alleviate metabolic burden in E. coli cells producing high levels of a recombinant protein.