General Introduction: Recombinant Protein Production and Purification of Insoluble Proteins

Abstract

Proteins are synthesized in heterologous systems because of the impossibility to obtain satisfactory yields from natural sources. The production of soluble and functional recombinant proteins is among the main goals in the biotechnological field. In this context, it is important to point out that under stress conditions, protein folding machinery is saturated and this promotes protein misfolding and, consequently, protein aggregation. Thus, the selection of the optimal expression organism and the most appropriate growth conditions to minimize the formation of insoluble proteins should be done according to the protein characteristics and downstream requirements. Escherichia coli is the most popular recombinant protein expression system despite the great development achieved so far by eukaryotic expression systems. Besides, other prokaryotic expression systems, such as lactic acid bacteria and psychrophilic bacteria, are gaining interest in this field. However, it is worth mentioning that prokaryotic expression system poses, in many cases, severe restrictions for a successful heterologous protein production. Thus, eukaryotic systems such as mammalian cells, insect cells, yeast, filamentous fungus, and microalgae are an interesting alternative for the production of these difficult-to-express proteins.

The E. coli pET expression system revisited—mechanistic correlation between glucose and lactose uptake

“…E. coli can be cultivated on inexpensive media to high cell densities and has a high growth rate; its genetics are very well characterized and an increasingly large number of cloning vectors and mutant host strains are available (e.g., Baeshen et al 2015 ; Rosano and Ceccarelli 2014 ). The E. coli strain BL21(DE3) and its derivatives are by far the most used E. coli strains for recombinant protein production as they exhibit several biotechnological advantages compared to other E. coli strains, such as low acetate yield, high biomass yield, and reduced expression of proteases (Choi et al 2006 ; Ferrer-Miralles et al 2015 ; Rosano and Ceccarelli 2014 ). Usually, the well-known pET expression system is used in combination with E. coli BL21(DE3) (Studier and Moffatt 1986 ).…”

Heterologous Expression, Purification and Immunoreactivity of the Antigen 5 from Polybia paulista Wasp Venom

“…Recombinant production of antigen 5 often resulted in high levels of protein yields, regardless the cells system used for expression. Although E. coli remains a highly popular system for heterologous expression, its use is hampered by protein aggregation and miss folding, which finally affect the downstream use of the product [ 30 ]. Considering the results previously obtained by our group for the expression of P. paulista major allergens, Poly p 1 [ 12 ] and Poly p 2 [ 13 ] in E. coli which resulted in insoluble products, we aimed the expression of Poly p 5 in the eukaryotic system P. pastoris .…”

Gene cloning, expression and polyclonal antibody preparation of Rab3A for protein interaction analysis

“…Generally speaking, owing to the continuous development and improvement of PCR technique, the cloning of a gene with known nucleotide sequence is relatively easy. Comparatively, protein expression and purification are more difficult because they involve many issues, such as the selection of optimal expression organism, high efficiency expression vector, most appropriate growth condition, efficient purification strategy, etc., which in turn depend on the protein characteristics and downstream requirements (Ferrer-Miralles et al 2015 ). The production of soluble and functional recombinant proteins is among the goals in the heterologous expression field.…”