Genetic design for facilitated production and recovery of recombinant proteins in Escherichia coli

Abstract

Genetic strategies have been used for more than two decades to improve bacterial bioprocesses and to simplify recovery procedures. Such strategies include the design of efficient expression vectors and the improvement of bacterial production strains in different ways, e.g. by deletion of protease genes or engineering for overexpression of rare-codon tRNAs, foldases or chaperones. Gene multimerization is another such principle that has proved beneficial to improve production yields. Genetic strategies have furthermore been exploited to facilitate recovery processes by adapting the product for a particular purification principle. In this area, affinity fusions have been commonly used, but other principles, such as modified isoelectric point (pI) or hydrophobic properties have also been successfully investigated. A recent drastic step forward in the use of gene technology to improve recovery processes for recombinant proteins is the introduction of combinatorial protein engineering to generate tailor-made product-specific affinity ligands. This strategy, which allows efficient recovery of a recombinant protein in its native form, is likely to be increasingly used also in industrial-scale bioprocesses, since novel protein ligands have been described that can be sanitized using common industrial cleaning-in-place procedures. The examples presented in this review make it evident that genetic strategies will be of utmost importance in the future for facilitating production and recovery of recombinant proteins.

Optimization Of Expression Conditions Of The Acetylesterase CE16 From Hypocrea Jecorina Encoded By A Synthetic Gene And Expressed In Escherichia coli Cells

“…2). Codon usage was optimized for the expression in E. coli cells in order to overcome problems originating from the differences between the eukaryotic gene and the prokaryotic host organism (JONASSON et al, 2002). The prepared gene was inserted into the pET21a vector with C-terminal His6-tag sequence and a gene responsible for ampicillin resistance (Fig.…”

New Generation Expression Host System- Aiming high for commercial production of recombinant protein

“…In addition to codon bias and lack of post translational modification machinery, many proteins expressed in E. coli got accumulated intracellular as inactive inclusion bodies [34,35]. Expressed protein in the form of inclusion bodies further needs protein renaturation and refolding to retain biological activity, all these transformations are often complicated and costly while running denaturation and refolding process [36].…”

Exploitation of prokaryotic expression systems based on the salicylate-dependent control circuit encompassing nahR/Psal

“…The main characteristic of an attenuation system that makes it attractive as a complementary regulatory circuit in expression vectors is that it prevents transcription elongation regardless from (1) undesired basal transcription from the regulated promoter of the expression system or (2) spurious initiation of the bacterial Thus, spurious promoter activity, plasmid read-through or cryptic initiation signals clearly raises basal expression levels of heterologous genes in high-copy number expression vectors, 37 thereby favoring the appearance of expression-down phenotypes in the case of gene expression that decreases the growth rate of the host strain. 29,37,38 To overcome this problem different approaches have been developed to tighten the control of gene expression, such as the reduction of gene dosage either by using low-copy number plasmids or by chromosome integration.…”

“…29,37,38 To overcome this problem different approaches have been developed to tighten the control of gene expression, such as the reduction of gene dosage either by using low-copy number plasmids or by chromosome integration. 29,39,40 However, these modified systems do not sustain a high level of gene expression.…”