1. Construction of expression cells
The process of obtaining recombinant protein drugs from their proteins belongs to genetic engineering pharmacy; if yeast cells in prokaryotic cells or eukaryote are used as hosts for protein expression, this technique belongs to fermentation engineering; If mammalian cells or other animal and plant cell culture expression is used, it can be summarized into the field of cell engineering and pharmaceutical; if the expression product is an enzyme, the process of preparing the enzyme can also be included in the research field of enzyme engineering; if the expression product is antibody, it can be counted as antibody engineering, etc. In any case, the core is genetic recombination, engineering cell construction, and the final product is protein, so they are collectively called recombinant protein drugs.
(1). Key elements for construction of expressing host cell
A gene is a DNA or RNA sequence that encodes a biologically functional molecule. In the process of gene expression, DNA is first copied into RNA, and RNA can be translated into a protein directly or as an intermediate template to function.
The target genes are prokaryotic genes and eukaryotic genes. Mammalian genes are generally composed of introns and exos, where exons are structural genes of functional proteins, are sequences that occur in mature RNA, also known as expression sequences; introns are the parts that lose expression function, which usually appears in the precursor RNA, and is cleaved off before translation.
In cell biology, a group of allogeneic cells with a common ancestor is called a clone; genetically, the same phenomenon that the genetic information of a living being is identical to the genetic information of its parent is also called cloning; in molecular biology, cloning refers to the process or method of replication, amplification of a particular molecule.
The gene must be expressed in a certain host cell, which must first be recombined with a vector to be introduced into the host cell for cloning, preservation and expression. This vector is an important tool for carrying genes into the host cell. Generally vectors has the following characteristics:
1). Can be independently replicated in the host cell without affecting the normal physiological activity of the host cell;
2). There are certain selection marks for easy identification and screening;
3). It is possible to insert a large molecular weight foreign DNA sequence without affecting its own replication;
4). There are suitable restriction sites for cloning.
According to the above characteristics, people obtain some natural carriers from nature, and use DNA recombination technology to optimize and reorganize, and construct many carriers with specific properties. In the development of molecular cloning, a variety of vectors, such as plasmid, λ phage, filamentous phage, phagemid or phasmid, cosmid, charomid, a yeast cloning vector, a plant cloning vector, and an animal cell cloning vector have been used. Some plasmids can express proteins in prokaryotic or eukaryotic cells, which is often referred to as shuttle plasmids. These vectors vary in size, ranging from a few kb to tens of kb. There are two types of tight and relaxed type. The tight type can only produce several copies in the cell, the molecular weight is large, and its replication is often accompanied by cell chromosome replication; the relaxed type may produce dozens of copies, and other properties are opposite to the tight type. Vectors are incompatible to each other. In the same host cell, there are often no multiple carriers at the same time. Even if there are more than two carriers in a period of time, after some time, one carrier will compete to gain advantages, and the other carriers will gradually disappear. Only vectors that are not in the same incompatible group can coexist in a host cell.
In the process of genetic recombination, depending on the main role of the vector, the vector is often divided into a cloning vector and an expression vector. The cloning vector is mainly used for gene preservation, cloning, or construction of a gene bank; the expression vector is mainly used for expressing a protein of interest in a host cell, or constructing a gene bank for expression. Most vectors have the dual function of a cloning vector and an expression vector.
Vector for expression of a recombinant protein gene, usually has the expression structure of a promoter, a multiple clone site (MCS), an antibiotic resistance gene, a terminator, a marker molecule, and other factors such as a regulatory factor, enhancers, which contribute to soluble expression. For the prokaryotic expression vector, by changing the temperature, the partial ion concentration in the medium, or administering a special inducer, the promoter can be activated, or the inhibition of the promoter can be released, and the expression process of the target protein can be initiated.
The most widely used is the Lac promoter, which is a nucleotide sequence in a lactose operon on a DNA molecule. The lactose operon consists of three genes, such as a repressor gene (LacI), a promoter, and an operator, and a gene encoding three enzymes involved in lactose utilization (LacZ, LacY, LacA). The addition of lactose or an analog thereof, such as isopropylthiogalactoside (IPTG), will bind to the repressor, alter the configuration of the Lac promoter, de-repress, and initiate the transcription process. There are other commonly used promoters, such as the T7 promoter, the trp promoter, the Tac promoter, the lPL promoter, and the like. For eukaryotic cells, a signal peptide is also present on the expression vector, and the anti-neo gene and the like for G418 screening can form a stable expression of engineering cells by transient expression or chromosomal gene transfer.
c. Host cell
As mentioned above, the commonly used host cells include prokaryotic cells and eukaryotic cells.
Prokaryotic cells generally use bacteria. Escherichia coli and Bacillus subtilis are the most commonly used, of which E. coli is the most widely used. The most commonly used expression strains for Escherichia coli are BL21 (DE3), DH5α, JM109 and the like. The prokaryotic expression system has many advantages such as stable expression, simple construction, and low cost. The biggest problem is that the foreign protein lacks glycosylation and phosphorylation after transcription, and it cannot form complex disulfide bonds.
Eukaryotic cells mainly include yeast cells, insect cells, mammalian cells, and plant cells. Yeast cells can undergo a degree of glycosylation modification, but the class of sugars differs from mammalian cells. In addition, there is a problem of expression stability during the induction of expression. Insect cell expression mainly corresponds to the insect baculovirus vector and has the advantage of high expression level. However, due to the potential safety of the insect baculovirus, there is currently no marketed product using this expression system. Mammalian cells are the mainstream expression system in the current production process of recombinant protein drugs, and have the closest structure and modification characteristics to human natural proteins. Commonly used mammalian cell expression systems include Chinese hamster ovary cells (CHO), human embryo kidney 293 (HEK293), baby hamster kidney cells (BHK), and human fibrosarcoma, cells (HT-1080), etc. Plant cells have a short expression time and have not yet been developed for clinical use. As for the process technology of transgenic plants and animals for the preparation of recombinant protein drugs, there are some successful research and development results, or successful development of drugs, in view of the accuracy and expression level of gene mapping, can not be used as the mainstream technology for drug preparation.
To be continued in Part Three…