First, the experimental design?
We analyzed the target gene sequence provided by our customers. The whole protein sequence is hydrophilic and there is no signal peptide sequence, so our design idea is as follows:
1. 1. Using the protein sequence translated from the customer's gene sequence as the source template, a group of gene sequences most suitable for HEK293 cell line were obtained through codon optimization.
1.2. It is suggested that customers use the autonomous carrier pAZ-V5 of Zhongding Bio to carry out experiments. The V5 signal peptide sequence of the vector is helpful for protein to secrete and express outside the cell. In order to keep the N-terminal activity of the protein, we added 6XHis-Tag to the C-terminal, which is convenient for the detection and affinity purification of the recombinant protein.
According to the above design idea, pAZ-V5 expression plasmid was constructed by gene synthesis. After sequencing and restriction enzyme digestion, the plasmid without endotoxin was selected. 200ml HEK293 cells were transfected, and the protein expression was detected by western blot, and then more than 80% of the target protein was obtained by Ni- resin affinity purification.
Second, reagents and consumables
Company name company name
High glucose in DMEM, low glucose in DMEM, fetal bovine serum, trypsin, opti SFM, transfection reagent Invitrogen(Gibco) endotoxin-free plasmid extraction kit, LAL endotoxin detection reagent, basic chemical reagent, low melting point agarose Sigma Company.
Extraction kit Axygen PCR kit IO-RAD company
Six-well plate, cell culture bottle, centrifugal tube PCR reaction tube Fisher Company
DNA gel purification kit, plasmid extraction kit AXYGEN company agarose Shanghai gene company.
Other reagents of Neutral Red Blue Sky Biological Company are analytically pure domestic.
Second, the main experimental instruments
Instrument Name Company Instrument Name Company Instrument Name Company
Allegra 2 1R Desktop High-speed Freezing Centrifuge BECKMAN Desktop High-speed Centrifuge SORVAL Germany Mini Protean II Vertical Plate Electrophoresis System, Gel Doc2000 Imaging System and Horizontal Electrophoresis System BIO-RAD USA.
The PTC-200 gene amplifier 320-S pH meter of MJ Research Company of the United States, the AR5 120 electronic balance of mettler toledo Company of the United States, and AHOM Company of the United States.
MultiTemp III constant temperature water bath pot, HoferμV-25 ultraviolet emitter American Amersham Pharmacia snowflake ice maker Japanese Sanyo JY92-2D ultrasonic cell pulverizer China Xinzhi Keqi Institute.
Protein nucleic acid detector Nanjing University Puyang Scientific Instrument Factory NANODROP2000 Thermoelectric Company Clean Worktable China Su Jing Group
Fourthly, the analysis of the nature of protein.
4. 1 Original gene sequence provided by customers
Gtggggtgct * * * * NNNNNNNNNNNNNNNNNNNN-involves customer research content, and will not display-NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN * * * * CTTCTCCCTT.
4.2 The encoded protein sequence is as follows (theoretical molecular weight 4 1. 18KD, theoretical isoelectric point PI6.73).
GCSVDFSK * * * * * nnnnnnnnnnnnnnn-nnnnn nnn nnn nnn nnn nnn * * * * * TagSTDHMDHFSL。
4.3 Sequence analysis of rare codons is shown in the following figure (red: frequency of use is lower than 10%, gray: frequency of use is lower than 20%, and green is normal frequency codon).
4.4 Analysis of transmembrane and hydrophilicity of protein sequence
4.5 signal peptide prediction
To sum up:
1, the theoretical molecular weight of protein is 4 1KD, which belongs to the normal molecular weight range and is suitable for expression.
2. Based on the analysis of gene codon usage frequency, if 293 cells are used as the expression host, X codons with frequency less than 20% and X codons with frequency less than 10% are used in the gene sequence, and the target gene is obtained through gene synthesis after optimization.
3. Through the analysis of the transmembrane structure and hydrophobicity of the protein, the protein is hydrophilic as a whole, but after 473AA, the protein has a typical transmembrane structure, so it is suggested to remove it before expression to improve the success rate.
4. The target protein itself has no signal peptide, and it is convenient to secrete and express the protein by adding GP67 signal peptide. For the convenience of purification, we can consider adding His tag to C-terminal.
Verb (abbreviation of verb) experimental method and results
5. Construction of1PAZ-V5 expression plasmid
The target gene was synthesized by PAS (Precise Synthesis Based on PCR), and was ligated between Afl II and Xba I of pAZ-V5 vector by double digestion. The recombinant plasmid was transferred to the TOP 10 clone strain, and the positive clone was selected for sequencing. The sequencing results are spliced as follows, and the single dotted line region is the gene region of the target gene. The purple region is the restriction site, the yellow region is the v5 signal peptide, and the green region is the 6XHis tag.
This is a good example, which can be said to be a good example.
5.2 Use Chromas software to view the sequencing peak file. Examples of intercepting some sequences are as follows:
5.3 pAZ-V5 carrier schematic diagram is as follows:
Optimization of expression conditions
For more service descriptions, please consult Zhongding Bio-official website.