Microbial Biotechnology Protein Production.pptx

Protein production techniques

1. Microbial Biotechnology: Protein Production in Bacteria and Yeasts Dr G.O Oyetibo Dept of Microbiology, University of Lagos, Nigeria

2. Recombinant proteins It has been utilized as tools for cellular and molecular biology Unlimited accessibility of therapeutic and prophylactic proteins to saving lives unlike before the era of modern biotechnology when a very small amounts from unsafe sources were obtained. More than 75 recombinant proteins are currently utilized as pharmaceuticals, and more than 360 new medicines based on recombinant proteins are under development It also extends to the development of bioinsecticides , diagnostic kits, enzymes with numerous applications, and bioremediation processes among others.

3. Problems encountered during recombinant protein production Loss of expression of the gene of interest due to structural changes in the recombinant gene, or disappearance of the gene from host cells. This occurs at three alternative locations of the gene of interest In plasmids as plasmid loss, low-copy-number plasmids, high copy number, plasmid structural instability Chromosomal integration resulting in low production rate due to low copy number of recombinant gene Viral vectors

4. Problems encountered during recombinant protein production contd Posttranslational processing Folding, aggregation, and solubility. Foldases accelerate protein folding; chaperones prevent the fomation of non-native insoluble folding intermediates. Misfolded protein accumulate in intracellular aggregates called inclusion bodies. Aggregation protects proteins from proteolysis and can facilitate protein recovery by simply breaking the cells and centrifuging the inclusion bodies Proteolytic processing. Signal peptides, needed to direct proteins to the various cellular compartments, must be cleaved by signal peptidase complex to obtain a functional protein, otherwise it may result in protein aggregation and retention within incorrect compactments such as the endoplasmic reticulum Glycosylation . Involves tens of enzymes and substrates, and occur in the endoplasmic reticulum and Golgi apparatus of eukaryotes. In many cases, glycosylation determines protein stability, solubility, antigenicity , folding, localization, biological activity, and circulation half-life.

5. Bioengineering approaches to solve problems associated with heterologous gene expression Induction strategies, where recombinant genes can be placed under a variety of promoters. The promoter gene will determine whether gene expression is constitutive or inducible. Growth control, since growth rate affects several parameters that determine recombinant protein accumulation rate. Among these parameters are the percentage of substrate utilized for cellular maintenance, RNA polymerase activity, ribosome number, plasmid stability, plasmid copy number, plasmid multimerization , and the distribution of cells in the cell-cycle phases Bioreactor and operation strategies involving containment and the control of environmental parameters in predetermined values.

6. Post-translational modification of insulin. At the top, the ribosome translates a mRNA sequence into a protein, insulin, and passes the protein through the endoplasmic reticulum, where it is cut, folded and held in shape by disulfide (-S-S-) bonds. Then the protein passes through the G olgi apparatus, where it is packaged into a vesicle. In the vesicle, more parts are cut off, and it turns into mature insulin

7. Production of recombinant insulin in bacteria

8. Protein production in bacteria Notably Escherichia coli Merits Easy to grow to high cell densities (over 100 g/l) Has simple nutritional requirements that can be satisfied with fully defined simple media Rapid expression Ease of genome modifications inexpensive Drawbacks Usually not capable of efficiently producing very long or short proteins, although the successful expression of a 210 kDa protein has been achieved Proteolytic cleavage and disulfide bond formation seldom occur, and posttranslational modifications, including glycosylation , acylation , and isoprenylation , are not performed Bacteria possess pyrogens and endotoxins that must be totally eliminated from proteins to be injected in animals or humans Variability in the level of expression, protein solubility, and protein purifications Produce unglycosylated proteins Proteins produced as inclusion bodies, are inactive, require refolding

9. Bacteria The recombinant strain E. coli K12 is the most used Well-studied, documented and optimised as host for gene cloning High levels of homologous protein expression Relatively simple and inexpensive media High cell growth rates Formation of inclusion bodies or insoluble aggregates of proteins Difficulties in downstream processing; Presence of endotoxin molecule LPS in the membrane; Does not always recognise the upstream elements of recombinant genes; Limited capability to carry out PTMs (glycosylation) disadvantages advantages

10. Protein production from yeast Yeasts have been used since the early 1980s for the large-scale production of intracellular and extracellular proteins of human, animal, and plant origin Merits Used to synthesize life-saving drugs for pharmaceutical industry Used to unravel the complex regulatory phenomena at the heart of basic research

11. Suitability of yeasts for protein production Yeasts offer the ease of microbial growth and gene manipulation found in bacteria along with the eukaryotic environment Ability to perform many eukaryote-specific post-translational modifications, such as proteolytic processing, folding, disulphide bridge formation, and glycosylation . Relative to more complex eukaryotic expression systems like Chinese hamster ovary cells and baculovirus -infected cell lines, yeasts are economical, usually give higher yields, and are less demanding in terms of time and effort. Disadvantages to using yeasts for expression of some heterologous proteins Inability to perform certain complex post-translational modifications, such as prolyl hydroxylation and amidation , as well as some types of phosphorylation and glycosylation . Extracellular proteases are expressed N- glycosylation of proteins produced by yeasts are high-mannose (with more than 3 mannose residues) or hypermannose (more than 6 mannose residues) types, with terminal α -1,3 linkages. Such forms are very immunogenic to mammals.

12. Steps of recombinant protein production in yeast This involves four steps Cloning of a foreign protein-coding DNA sequence within an expression cassette containing a yeast promoter and transcriptional termination sequences; Transformation and stable maintenance of this DNA fusion in the host; Synthesis of the foreign protein under specified culture conditions; and Purification of the heterologous protein and comparison with its native counterpart

13. A regulatable promoter is used to drive foreign protein expression because, prior to induction, the ability to maintain cultures in an ‘expression off’ mode minimizes selection for non-expressing mutant cells during the cell growth phase. Such a selection can occur as a result of the added metabolic burden placed on cells expressing high levels of a foreign protein or the toxic effect of a foreign protein on the cells. Notable yeasts commonly used are methylotrophic yeast Pichia pastoris, Pichia methanolica , Candida boidnii , Hansenula polymorpha etc., lactose-utilizing Kluyveromyces lactis , starch-utilizing Schwanniomyces occidentalis , xylose utilizing Pichia stipitis , alkane and fatty acid utilizing Yarrowia lipolytica , and the baker’s yeast Saccharomyces cerevisiae.

14. Pichia pastoris as engineered system for heterologous protein expression It has been utilised to produce ~300 foreign proteins since 1984. Factors that make this system popular: The use of the alcohol oxidase I ( AOX1 ) promoter, one of the strongest most regulated promoters known The ability to stably integrate expression plasmids at specific sites in the P. pastoris genome in either single or multicopy ; The ability to culture strains in high density fermenters ; and Its ready availability as a kit from Invitrogen Corporation (Carlsbad, Ca, USA) The AOX1 promoter is tightly repressed by glucose and most other carbon sources but is induced >1000-fold in cells shifted to methanol as a sole carbon source. With this promoter, expression of recombinant proteins is highly repressed while cultures are grown to high density in glucose or glycerol, which prevents selection for non-expressing mutant cells. Cultures are then shifted to a methanol medium to induce rapid high-level expression.

15. Limitations to this system Although methanol (a petroleum byproduct) is essential for maximum induction of the AOX1 promoter, it is a potential fire hazard and may not be appropriate for the production of food products. Thus a strong promoter that do not require methanol for induction are needed. Eg , glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter provides a constitutively high level of expression on glucose, glycerol, and methanol media Methanol is toxic to humans, and must be thoroughly removed from the final product Methanol is also toxic to the cells. Thus specially designed methanol feeding strategies must be implemented to guarantee its continuous supply during the induction stage but avoiding its accumulation to inhibitory levels. It lacked moderately expressed promoters. The high level of expression provided by the AOX1, FLD1 and GAP promoters is toxic in some cases and may overwhelm the protein-handling machinery of the cell, causing a significant portion of the protein to be misfolded or unprocessed Existence of only a few selectable markers for P. pastoris transformation

16. Yeasts It’s considered as a GRAS ( generally regarded as safe) organism Well-characterised; Easy insertion of homologous DNA sequences at specific locations in the yeast genome; Being a eukaryotic, it’s able to carry out PTMs (post-translational modifications) of protein; Fermentation conditions are known for thousands years Cell growth is slower; Glycosylation pattern may be inappropriate or different from the one observed in native glycoprotein. disadvantages advantages