ABSTRACT
In the 1980s, Escherichia coli was the preferred host for heterologous protein expression owing to its capacity for rapid growth in complex media; well-studied genetics; rapid and direct transformation with foreign DNA; and easily scalable fermentation. Despite the relative ease of use of E. coli for achieving the high expression of many recombinant proteins, for some proteins, e.g., membrane proteins or proteins of eukaryotic origin, this approach can be rather ineffective. Another microorganism long-used and popular as an expression system is baker's yeast, Saccharomyces cerevisiae. In spite of a number of obvious advantages of these yeasts as host cells, there are some limitations on their use as expression systems, for example, inefficient secretion, misfolding, hyperglycosylation, and aberrant proteolytic processing of proteins. Over the past decade, nontraditional yeast species have been adapted to the role of alternative hosts for the production of recombinant proteins, e.g., Komagataella phaffii, Yarrowia lipolytica, and Schizosaccharomyces pombe. These yeast species' several physiological characteristics (that are different from those of S. cerevisiae), such as faster growth on cheap carbon sources and higher secretion capacity, make them practical alternative hosts for biotechnological purposes. Currently, the K. phaffii-based expression system is one of the most popular for the production of heterologous proteins. Along with the low secretion of endogenous proteins, K. phaffii efficiently produces and secretes heterologous proteins in high yields, thereby reducing the cost of purifying the latter. This review will discuss practical approaches and technological solutions for the efficient expression of recombinant proteins in K. phaffii, mainly based on the example of enzymes used for the feed industry.
ABSTRACT
Currently, HIV-1 CRF63_02A6 is the prevalent genetic variant of the HIV-infected subjects in the major part of the Siberian Federal District (Russia). The HIV-1 CRF63_02A6 R5-tropic pT11.17 and X4-tropic pMtBs.18 infectious molecular clones (IMCs) were constructed using the virus isolates recovered in 2015 and 2017 of male HIV-infected Russian residents (from Tomsk and Novosibirsk, respectively). Near full-length proviral HIV-1 sequences (9,644 and 9,748 bp) were subcloned in pBluescript II KS(-). The CRF63_02A6 IMC virions were obtained by transfecting HEK293T cells with the constructed plasmids and demonstrated a stable growth in peripheral blood mononuclear cell culture (p24 concentration increased >1,000-fold and the virus protein accumulation in culture liquid exceeded 100,000 pg/mL). The tropism of CRF63_02A6 IMCs was determined genotypically (using Geno2pheno) and phenotypically by cultivating the IMC virions in MT-2, U87-CD4-CCR5, and U87-CD4-CXCR4 cell cultures. The obtained HIV-1 CRF63_02A6 IMCs may be useful in basic and applied research.
