Production of Recombinant Proteins Protein Production in Prokaryotic Hosts Regulation of transcription Increasing translation efficiency Increasing protein stability Increasing protein secretion Facilitating protein purification DNA integration into the host chromosome Protein Production in Eukaryotic Hosts Posttranslational modification of eukaryotic proteins Eukaryotic expression systems (general features, yeast, baculovirus-insect cells, mammalian cells) Protein Engineering Directed mutagenesis Random mutagenesis Examples of protein engineering

Table 3.1 For many biotechnology applications, the primary objective is to produce high levels of a protein from a cloned gene expressed in a host organism. Each host organism, has specific advantages and disadvantages.

Protein Production in Prokaryotic Hosts Figure 3. 1 A strong E Protein Production in Prokaryotic Hosts Figure 3.1 A strong E. coli promoter with the -35 and and -10 boxes which bind to RNA polymerase to allow for transcription.

Table 3.2 Regulation of transcription

Table 3.3

Figure 3.6 The plasmid expression vector pKK233-2 uses the tac promoter (ptac), a ribosome binding sequence (rbs) to allow for translation, three restriction enzyme sites to clone a foreign gene, and two prokaryotic transcription termination sequences (T1 and T2).

Table 3.4

Table 3.4 (Continued)

Table 3.5 Increasing translation efficiency: Codon optimization can greatly enhance protein production.

Increasing Protein Stability By facilitating protein folding (e.g., low temperature or coexpress with genes encoding proteins that function in proper protein folding such as chaperones) By decreasing protein degradation (e.g., add specific amino acids to the N-terminus to reduce protease digestion or express the protein as a fusion protein) Figure 3.9

Increasing Protein Secretion Secretion into the periplasm by adding a bacterial signal peptide sequence to the N-terminus generally allows for easier protein purification and reduced protein degradation as there are fewer proteins and proteases in the extracellular environment. Figure 3.10 Protein secretion in bacteria. (A) Secretion pathway in Gram-negative bacteria like E. coli. B. Protein secretion in Gram-positive bacteria such as Lactococcus lactis.

Table 3.9 Facilitating protein purification

Figure 3.14 Purification of a fusion protein by immunoaffinity chromatography.

Figure 3.15 A proteolytic cleavage site can by genetically engineered into a fusion protein to facilitate release of a target protein from any affinity tag. Blood coagulation factor Xa is one such protease. Government agencies which regulate pharmaceutical proteins require proteins free of affinity tags.

Figure 3. 18 DNA integration into the host chromosome Figure 3.18 DNA integration into the host chromosome. A cloned gene can be integrated into a bacterial chromosomal site in order to reduce the metabolic load imposed on a bacteria by a plasmid vector.

Protein Production in Eukaryotic Hosts Unlike prokaryotes, eukaryotes carry out a number of posttranslational modifications (PTMs) of proteins, which are often critical for proper protein function These PTMs include: Glycosylation Processing of propeptides Phosphorylation Acetylation Methylation Hydroxylation Sulfation Acylation g-Carboxylation Myristoylation (C14 fatty acid addition) Palmitoylation (C16 fatty acid addition) Disulfide bond formation

Figure 3.22

Figure 3. 25 Generalized eukaryotic expression vector Figure 3.25 Generalized eukaryotic expression vector. p=eukaryotic promoter; MCS=multiple cloning site; t=eukaryotic transcription termination sequence; ESM=eukaryotic selectable marker; orieuk=eukaryotic origin or replication; oriE= E. coli origin of replication; Ampr gene=E. coli selectable marker

Table 3.10

Figure 3.28 Protein secretion pathway in eukaryotes requires a eukaryotic Signal Peptide Sequence.

Figure 3.29 Proper protein folding in the ER is important for protein function. Protein disulfide isomerase (PDI) and the molecular chaperone BiP helps with protein folding. Adapted from Gasser et al., Microb. Cell Fact. 7:11–29, 2008.

Figure 3. 31 Pichia pastoris (yeast) integrating expression vector Figure 3.31 Pichia pastoris (yeast) integrating expression vector. AOX=alcohol oxidase 1 gene

Figure 3. 34 Baculovirus-Insect Cell Expression Sytems Figure 3.34 Baculovirus-Insect Cell Expression Sytems. Baculoviruses are are double-stranded DNA viruses which infect arthopods (insects). A. Budded form of the baculovirus. B. Occluded form of the baculovirus.

Table 3.14

Figure 3. 40 Mammalian Cell Expression Systems Figure 3.40 Mammalian Cell Expression Systems. Generalized mammalian expression vector. P=eukaryotic promoter; I=intron (which enhances protein production); MCS=multiple cloning site; SMG=eukaryotic selectable marker gene; pa=polyadenylation addition sequence; TT=eukaryotic termination of transcription sequence.

Figure 3.41 Translational control elements for eukaryotic protein production, secretion, and purification. K=Kozak sequence [GCCGCC(A or G)CCAUGG]; S=signal peptide sequence; T=protein affinity tag; P=proteolytic cleavage site; SC=stop codon

Figure 3.47 Protein engineering to improve or enhance protein function can be done by directed mutagenesis (as shown here) or by random mutagenesis.

Table 3.17

Table 3.18