I. Purpose of Linearization

The main function of the vector is to carry the target gene into the host cell, and only when the vector is linearized can it complete recombination with the exogenous target gene to construct a recombinant plasmid.

Different from prokaryotic expression, in yeast expression, the plasmids or vectors used for transformation often do not contain the replication origin of the yeast itself, so if the cyclic recombinant plasmid is directly transferred into the yeast cells, it cannot exist stably, so it must be linearized and then integrated into the yeast chromosome in a homologous recombination manner so that the target gene can exist stably in the yeast cells.

II. Commonly Used Vectors in The Yeast System

Types of yeast vectors include yeast cloning vectors, yeast expression vectors, and yeast artificial chromosomes (YAC).

(1) Yeast cloning vectors: they do not contain yeast promoters and cannot express exogenous genes in yeast, and can be divided into two categories:

a. Yeast integrated plasmid (Yip): with a yeast URA3 marker gene and replication and reporter genes from E. coli. Due to homologous recombination between plasmid DNA and yeast genomic DNA, integrated replication of the plasmid can be detected in the transformed cells, and the transformants are stable, but the transformation rate is extremely low.

b. Yeast replicable plasmid (Yrp): This type of vector can replicate itself in yeast, mainly YRp, YEp, and YCp.

Yeast replicable plasmid (YRp): RNA+Yeast genetic marker+Yeast replicable sequence (ARS), can replicate in yeast, but the stability is poor.

Yeast add-on plasmid (YEp): Bacterial plasmid DNA + yeast marker + yeast 2 μM plasmid, free from nucleus and replicates autonomously, good stability. 2 μm plasmid contains autonomous replication initiation region (ori) and STB region, and the STB sequences can stabilize the plasmid in the donor cells.

Yeast Chromosome Attachment Plasmid (YCp): In addition to the replication initiation region, this plasmid contains a Saccharomyces cerevisiae chromosome attachment nematode (CEN), which is capable of replicating outside the chromosome, with a low but stable copy number, and is suitable for use as a subcloning vector and for the construction of Saccharomyces cerevisiae genomic DNA libraries.

YIp: carries a yeast marker gene;

YRp: Yeast replication sequence (ARS) is added to YIp;

YEp: Yeast 2μM plasmid added to YIp.

(2) Yeast expression vectors: Containing the yeast promoter, the vectors of the yeast expression system are generally shuttle plasmids that can replicate in Saccharomyces cerevisiae and E. coli.

The production of recombinant proteins in the Saccharomyces cerevisiae expression system can be accomplished using three types of vectors: the integrating plasmid (YIp), the add-on plasmid (YEp), and the filamentous plasmid (YCp). In unconventional yeast, plasmid selection is more limited. Although plasmids can be used in all three types of unconventional yeast, they tend to have low copy numbers and are prone to mutation. Therefore, genome integration remains the preferred option.

Two commonly used expression systems in Picot yeast include secreted expression vectors and non-secreted expression vectors

a. Picrosimus secretory expression vectors

The pPICZα A, B and C vectors are used to express and secrete recombinant proteins in Picot yeast and can be used in any Picot yeast strains, including X-33, SMD1168H and KM71H.

The pPICZα vector contains the following elements:

• AOX1 promoter, which tightly regulates the relevant genes to ensure methanol-induced expression

• alpha-factor secretion signal for recombinant protein secretion

• (bleomycin) gene for screening in E. coli and Pichia yeast

• C-terminal peptide containing the c-myc antigenic determinant cluster and (6xHis) tag for detection and purification of recombinant fusion proteins.

b. Pichia yeast non-secretory expression vectors:

The pGAPZ A, B, and C vectors (2.9 Kb) use the GAP promoter to stably express recombinant proteins in Pichia yeast. recombinant proteins from the GAP promoter are expressed at a slightly higher level than the AOX1 promoter. In contrast to the inducible promoter, the constitutive promoter can utilize more carbon sources to express exogenous proteins in Pichia yeast. The recombinant proteins are expressed as fusion proteins with a C-terminal His tag. screening markers for Zeocin can be used in both Baker's yeast and E. coli.

(3) Yeast Artificial Chromosome

Yeast Artificial Chromosome (YAC) is a carrier capable of cloning DNA fragments up to 400Kb in length, containing telomeres, mitotic sites and replication initiation sequences necessary for yeast cells, and is an object of genetic nature within the cell, easily stained with alkaline dyes to a darker color, hence the name.

Characteristics: Large capacity (50-1000kb), poor stability.

Function: used to construct genomic libraries

Essential elements for replication in yeast include the Autonomous Reproducing Sequence (ARS), the mitotic and meiotic functioning of the mitosis (CEN) and the two telomeres (TEL).

Telomere Elimination Sequence (TEL): a sequence positioned at the end of the chromosome that protects the thread-like DNA from degradation by intracellular nucleases to form a stable structure.

Centromere (CEN): A binding site for spindle filaments during mitosis that allows chromosomes to be correctly distributed to daughter cells during division. YAC serves to ensure that there is only one artificial chromosome in a cell. For example, pYAC4 uses the mitogen of the fourth chromosome of yeast.

Autoreplicative Sequence (ARS): A specialized sequence containing signals necessary for bidirectional DNA replication in yeast.

The most commonly used of the YAC vectors is pYAC4. Since yeast chromosomes are linear, they are also linear in their working state. However, to facilitate the preparation of YAC vectors, YAC vectors are present in a looped form with the addition of the replication elements and selection markers of ordinary E. coli plasmid vectors.

III. Yeast System Carrier Selection

Yeast expression products have two ways of intracellular expression and secretion to extracellular expression, which depends on the choice of expression vectors and whether the construction is with signal peptide or not, and suitable yeast protein expression vectors can be selected according to the localization and purpose of gene expression.

① Vectors for intracellular expression: mainly include pPIC3, pPICZ, pPSC3K, pHIL-D2, etc. These vectors express the target gene in the cell. These vectors express the target genes in the intracellular area, which can avoid the glycosylation of yeast, and are suitable for non-glycosylated proteins that are usually expressed in the cytoplasm or do not contain -S-S-, which have a higher level of expression than those secreted from the extracellular area, but the purification is relatively complicated.

② Vectors secreted to extracellular expression: pPIC9, pHIL-S1, pYAM75P, etc. Yeast itself secretes few exogenous proteins, and secretion of exogenous proteins into the extracellular area is conducive to the purification and accumulation of the target proteins. The commonly used secretion signal sequence consists of 89 amino acids guided by the α mating factor.

③ Vectors for multi-copy insertion expression: pPIC9K, pPIC3.5K. In some cases, multi-copy integration of recombinant genes can increase protein expression.

IV. Linearization Verification and Interpretation of Results

Linearized yeast before and after run comparison electrophoresis. The cut strip should run slower (4-7) and the intact plasmid runs in front (1-3).

Figure 1 Carrier Linearization Verification Diagram

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