Practical Methods for Expression of Recombinant Protein in the Pichia pastoris System

Abstract

One of the most critical challenges of genetic engineering is the expression of recombinant proteins using biological expression systems. Nowadays, different expression systems from bacteria to mammalian tissue culture cells are available for the production of recombinant proteins for medical and industrial purposes. Among various choices, yeast expression systems such as Pichia pastoris are promising candidates. The P. pastoris expression system is a standard tool for the production of biopharmaceuticals and industrial enzymes. It is also considered a unique host for the expression of subunit vaccines which could significantly affect the growing market of medical biotechnology. Using P. pastoris as an expression system for heterologous proteins, this article provides detailed basic protocols for cloning of a recombinant cassette into a suitable expression vector, the transformation of foreign vector DNAs into the yeast by electroporation, and expression and purification of desired recombinant protein. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Cloning of a recombinant cassette into a suitable expression vector Basic Protocol 2: Transformation of P. pastoris and selection of transformants Basic Protocol 3: Optimization and large‐scale expression of recombinant proteins Basic Protocol 4: Purification of recombinant proteins

In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

“…We mentioned earlier (section ) the first reports from Brindle and colleagues using heterologous expression and 19 F-tryptophan incorporation in S. cerevisiae . , Recombinant expression in E. coli took over in the 2000s (section ), before yeasts were reused for in-cell structural NMR purposes a few years ago. These present interesting features: (i) their ease of genetic manipulation, (ii) their importance in biopharmaceutical protein production, − (iii) their capacity to integrate the cost-effective ammonium and various carbohydrates as sole source of nitrogen and carbon for isotope labeling, (iv) their numerous amino acid auxotroph strains, and (v) their established impact in the field of protein:protein interactions discovery, with the so-called yeast two hybrid technique . In-cell NMR could provide the atomic-scale description counterpart of the protein:protein binding revealed by yeast two hybrid.…”

Recombinant Thaumatin-Like Protein (rTLP) and Chitinase (rCHI) from Vitis vinifera as Models for Wine Haze Formation

“…For that, yeast cells were: (1) cultivated in 100 mL of yeast-peptone-dextrose (YPD) liquid medium (at 30 °C and 200 rpm, until the OD 600 reached 1.2); (2) harvested by centrifugation (4000× g , 20 min, 4 °C); (3) resuspended in 250 mL ice-cold water; (4) centrifuged again (4000× g , 20 min, 4 °C); and (5) finally resuspended in 1 mL of ice-cold 1 M sorbitol. Electroporation was performed by adding 10 µL (1 µg/µL) of the linearized DNA plasmid solution to 80 µL of the cell suspensions in chilled sterile cuvettes in which an electroporator (Eporator ® , Eppendorf SE, Hamburg, Germany) was inserted following standard protocols [ 49 ].…”

Komagataella phaffii as a Platform for Heterologous Expression of Enzymes Used for Industry

“…Along with a low secretion of endogenous proteins, K. phaffii efficiently and with high yields produces and secretes heterologous proteins, thereby reducing the cost of purifying the latter [ 28 , 29 , 30 ]. To date, practical approaches have been developed and technological solutions have been described for the efficient expression of recombinant proteins in K. phaffii [ 31 , 32 ].…”

“…When constitutive promoters are utilized, cultivation is facilitated, which in this case does not require a change of the carbon source, and the product is quickly synthesized; however, the high metabolic load on the cells in their growth phase will reduce their viability and is not suitable for the production of proteins toxic to yeast cells [ 72 , 73 ]. To date, a number of studies and reviews are available about using different promoters for heterologous expression, e.g., this study [ 32 ].…”