# Patent

## Title
Auto-developing and regularly-weighted protein molecular weight marker kit and method for preparing the same

## Abstract

The present invention provides an auto-developing and regularly-weighted protein molecular weight marker kit, comprising: (a) a plurality of recombinant proteins having formula (I),

(B)m-A-(C)n  (I),

    wherein A is a polypeptide of SEQ ID NO: 1, B and C are independently any mutually identical or different polypeptides with the value of molecular weight being a multiple of 5, and m and n is independently 0 or any integer larger than 0; and (b) one or more solvents for stabilizing the recombinant proteins. The present invention also provides a method for preparing the auto-developing and regularly-weighted protein molecular weight marker kit.

## Background
FIELD OF THE INVENTION
The present invention relates to a regularly-weighted protein molecular weight marker kit comprising 14-tag, which can be further used as a positive control of Western blotting.
BACKGROUND OF THE INVENTION
Protein markers play a crucial role in proteomics research with the coming of post-genome era. However, protein markers nowadays still have many restrictions and inconveniences. For example, (1) currently, the protein markers have to be copied manually from nitrocellulose paper to film instead of developing directly in the film during western blotting; (2) the common pre-stain protein markers sold on market have low-accuracy since the staining results in heterogeneity and electricity alteration; and (3) the current protein marker kits use known proteins as markers, yet their molecular weights are fixed and irregular. Therefore, there is a need for developing an auto-developing and regularly weighted protein molecular weight marker for Western blot to solve the problems encountered in proteomics research.
There are presently various types of protein markers for electrophoresis and Western blotting, and most of them are pre-stain markers. For instance, the multicolored protein marker is known for its colorful marker that enables easy observation, but the low-accuracy problem is still unsolved.
Chang et al. used a set of green fluorescent protein (GFP) fused proteins to construct dye-free protein molecular weight markers, which can emit fluorescence and present bands as regular as a ladder (Chang M., Hsu H. Y. and Lee H. J. Dye-free protein molecular weight markers. Electrophoresis, 26: 3062-68, 2005). Although the markers are convenience, they cannot be heated since GFP would be denatured and lose function. Without heating, however, the markers cannot be denatured thoroughly. Thus, the low-accuracy problem still remains.
Biotinylated protein markers are also available. These markers are dye-free but additional biotin label is required. Moreover, in order to be detected by color reaction, there is a need of labeling with HRP-conjugated anti-biotin antibody or HRP-conjugated avidin, which causes many inconveniences. In addition, biotin labeling may also alter the electric charge of protein marker and result in inaccuracy.
There are products of HIS-tag, S-tag or E-tag-fused protein markers as well. When using HIS-tag, S-tag, or E-tag antibodies to carry out development, the protein markers would auto-develop on film simultaneously. Those protein markers are not popular for the reason that the color presents simultaneously only when HIS-tag, S-tag, or E-tag antibodies is used to monitor protein expression. Otherwise, adding HRP-conjugated HIS-tag, S-tag, or E-tag antibodies is needed to activate the color reaction, which makes the procedure quite troublesome.



BRIEF DESCRIPTION OF THE DRAWINGS
The invention can be more fully understood by reading the subsequent detailed descriptions and examples with references made to the accompanying drawing, wherein:
FIG. 1 shows the schematic representation of the 14-tag protein marker of the present invention, which is auto-developed and regular molecular weight. The numbers on the left side of the figure represent molecular weight. In order to form the ladder-like 14-tag protein marker with regular molecular weight, the 14 tags is used as basic recognition unit and connected with GST (glutathione-S-transferase), MBP (maltose-binding protein), TRX (thioredoxin) and/or 5.0 kDa artificial peptides. When detecting target protein with anti-tag antibodies, the protein marker of present invention is recognized at the same time and developed on film.
FIGS. 2 A and 2B indicate the restriction enzyme maps of the recombinant protein markers. Constructing the restriction enzyme map in advance could not only decrease burdens of gene cloning for adding molecular weight but also prevent repetition of cutting sites. The numbers on the left side represent molecular weight; {circle around (s)} represents stop codon.
FIG. 3 shows (A) gene cloning design of basic recognition unit of 14-tag recombinant protein markers, GST, MBP, and TRX; and (B-C) size checking result of 14-tag, GST, MBP, and TRX by restriction enzyme digestion and agarose gel electrophoresis. (A) The schematic diagram of assembly polymerase chain reaction (PCR). 10 primers were designed, and 18 bp at 3′-end of one primer is complementary to the 5′-end of the next primer. Those primers were annealed into a linear sequence with restriction enzyme cutting sites at each end. (B) The size of assembly PCR product is confirmed about 409 bp by argarose gel electrophoresis. (C) The size of GST, after PCR amplification, insertion into pBlunt plasmid and restriction enzyme digestion, is confirmed about 645 bp. (D) The size of MBP and TRX, after PCR amplification, insertion into pBlunt plasmid and restriction enzyme digestion, are confirmed about 1134 bp and 300 bp respectively. M indicates protein molecular weight marker.
FIG. 4 shows the results that confirm 14-tag basic recognition unit expression. Western blotting is used to prove that the basic recognition unit (20 kDa) can be recognized by different antibodies and develop on film. The results confirm the feasibility of strategy of present invention since the basic recognition unit can be recognized by anti-HA antibody, anti-flag antibody, anti-cMyc antibody, anti-V5 antibody, and etc.
FIGS. 5A-F show the gene cloning result of 30-100 kDa protein markers. After assembling of 30-100 kDa protein markers, the size of each target fragments are confirmed 800, 1447, 1489, 1447, 2565, and 2544 bp by restriction enzymes, respectively. M indicates protein molecular weight marker.
FIGS. 6A-F show the gene cloning results of 25 and 120-240 kDa protein markers. After assembling of 25 and 120-240 kDa protein markers, the size of each target fragments are confirmed 705, 2173, 7565, 3253, 2181, and 2187 bp by restriction enzymes respectively. (+) indicates positive control; and M indicates protein molecular weight marker.
FIG. 7 shows the result of SDS-PAGE gel electrophoresis for analyzing molecular markers with different molecular weights.
FIG. 8 shows results of (A) anti-Myc antibody or (B-C) anti-V5 antibody as primary antibody for recognizing protein markers with different molecular weights. M indicates protein marker; P indicates positive control; and N indicates negative control. The unit of protein molecular weight is kDa.
FIG. 9 shows the recognition results of protein markers with different molecular weights by (A) anti-His antibody (1:200 dilution); (B) anti-S antibody (1:4000 dilution); (C) anti-V5 antibody (1:4000 dilution); and (D) anti-Myc antibody (1:7500 dilution) after adjustment. The 15 kDa to 100 kDa recombinant protein markers can be developed clearly and obtain similar results regardless of using what kinds of antibody. Images in FIGS. 9 A, B and D are captured by chemiluminescent detection system (Bio-rad). M indicates protein marker. The unit of protein molecular weight is kDa.
FIG. 10 shows the recognition results of protein markers with different molecular weights using (A) anti-Myc antibody (1:7500 dilution); (B) anti-HA antibody (1:4000 dilution); and (C) anti-flag antibody (1:7500 dilution) after combination and adjustment. The 15 kDa to 100 kDa recombinant protein markers can be developed clearly and obtain similar results regardless of using what kinds of antibody. Images in FIGS. 10 A to C are captured by chemiluminescent detection system (Bio-rad). M indicates protein marker. The unit of protein molecular weight is kDa.
FIG. 11 shows the linear regression curves of log value of molecular weight versus relative mobility measured from FIG. 9. The vertical axis indicates log value of molecular weight of protein marker; and the horizontal axis indicates relative mobility (mm). The equation of regression line can be represented as y=−0.0345x+2.08 and the obtained R2 value is 0.9821.



SUMMARY OF THE INVENTION
The present invention provides an auto-developing and regularly-weighted protein molecular weight marker kit, which comprises: (a) a plurality of recombinant proteins having formula (I),

(B)m-A-(C)n  (I),

wherein A is a polypeptide of SEQ ID NO: 1, B and C are independently any mutually identical or different polypeptides with molecular weight being about a multiple of 5, and m and n is independently 0 or any integer larger than 0; and (b) one or more solvents for stabilizing the recombinant proteins. The present invention also provides a method for preparing the auto-developing protein marker kit with regular molecular weight comprising: (a) constructing a plurality of nucleotide sequences encoding recombinant protein having formula (I) into DNA plasmids independently, and obtain a recombinant protein expressing vector,

(B)m-A-(C)n  (I),

wherein A is a polypeptide of SEQ ID NO: 1, B and C are independently any mutually identical or different polypeptides with molecular weight being about a multiple of 5, and m and n is independently 0 or any integer larger than 0; (b) transforming the recombinant protein expressing vector into competent cells; (c) selecting the competent cells carried the recombinant protein expressing vector; (d) administering the competent cell carried the recombinant protein expressing vector with inducer to induce expression of recombinant proteins; and (e) extracting recombinant proteins with different molecular weights independently, and mixing the plurality of recombinant proteins with one or more solvents for stabilizing recombinant proteins.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, the term “a plurality of” is employed to describe the number of elements and components of the present invention. This description should be read to more than one unless it is obvious that it is meant otherwise.
As used herein, the term “a” or “an” is employed to describe elements and components of the invention. This is done merely for convenience and to give a general sense of the invention. This description should be read to include one or at least one and the singular also includes the plural unless it is obvious that it is meant otherwise.
As used herein, the term “or” is employed to describe “and/or”.
The unit of molecular weight of polypeptide is kDa or kDa in the present invention.
In order to improve the flaws of above-mentioned well known technology, the present invention provides an auto-developing and regularly-weighted protein molecular weight marker kit, which comprises: (a) a plurality of recombinant proteins having formula (I),

(B)m-A-(C)n  (I),

wherein A is a polypeptide of SEQ ID NO: 1, B and C are independently any mutually identical or different polypeptides with the value of molecular weight being a multiple of 5, and m and n is independently 0 or any integer larger than 0; and (b) one or more solvents for stabilizing the recombinant proteins.
In which, the A of formula (I) is selected from one or more amino acid sequences of commercial tags and arranged properly. In some embodiments, the A is selected from the group consisting of His6-tag, HA-tag, T7-tag, E-tag, VSV-g-tag, V5-tag, AU5-tag, S-tag, HSV-tag, FLAG-tag, Lumio-tag, Glu-Glu-tag, cMyc-tag, and AU1-tag. The amino acid sequences and encoding DNA sequences thereof are shown in Table 1.







TABLE 1







The amino acid sequences of 14 tags and the encoding DNA sequences thereof









Tag
Amino acid sequence
DNA sequence





His6
SEQ ID NO: 19
SEQ ID NO: 20



HHHHHH
CATCATCATCATCATCAT


 


cMyc
SEQ ID NO: 21
SEQ ID NO: 22



EQKLISEEDL
GAACAAAAACTCATCTCAGAAGAGGATCTG


 


HA
SEQ ID NO: 23
SEQ ID NO: 24



YPYDVPDYA
TATCCATATGATGTTCCAGATTATGCT


 


S
SEQ ID NO: 25
SEQ ID NO: 26



KETAAAKFERQHMDS
AAAGAAACCGCTGCTGCTAAATTCGAACGC




CAGCACATGGACAGC


 


FLAG
SEQ ID NO: 27
SEQ ID NO: 28



DYKDADDDK
GATTACAAGGATGACGACGATAAG


 


V5
SEQ ID NO: 29
SEQ ID NO: 30



GKPIPNPLLGLDST
GGTAAGCCTATCCCTAACCCTCTCCTCGGTC




TCGATTCTACG


 


T7
SEQ ID NO: 31
SEQ ID NO: 68



MASMTGGQQMG
ATGGCTAGCATGACTGGTGGACAGCAAATG




GGT


 


E
SEQ ID NO: 32
SEQ ID NO: 33



GAPVPYPDPLEPR
GGTGCGCCGGTGCCGTATCCGGATCCGCTGG




AACCGCGT


 


VSV-g
SEQ ID NO: 34
SEQ ID NO: 35



YTDIEMNRLGK
TACACCGACATCGAGATGAACCGGTTGGGC




AAG


 


HSV
SEQ ID NO: 36
SEQ ID NO: 37



SQPELAPEDPED
AGCCAGCCAGAACTCGCTCCTGAAGACCCA




GAGGAT


 


Lumio
SEQ ID NO: 38
SEQ ID NO: 39



CCPGCC
TGTTGTCCTGGCTGTTGC


 


Glu-Glu
SEQ ID NO: 40
SEQ ID NO: 41



CEEEEYMPME
TGTGAAGAAGAAGAATACATGCCGATGGAA


 


AU1
SEQ ID NO: 42
SEQ ID NO: 43



DTYRYI
GACACCTACCGTTACATC


 


AU5
SEQ ID NO: 44
SEQ ID NO: 45



TDFYLK
ACCGACTTCTACCTGAAG









In some embodiments, under the premise without decreasing the developing effect of Western blotting, the arrangement and construction strategy of tags assembling A can be adjusted. Such adjustments include but are not limited to:

    
    
        (1) in order to achieve the goal of assembling regularly-weighted protein molecular weight marker, such as forming the ladder-like protein molecular weight marker, a tag is allowed to share one or more amino acids to the next tag to adjust the molecular weight;
        (2) in order to prevent particular restriction enzyme digestion in the sequences or to meet codon usage bias of cells expressing the recombinant proteins, one or more bases of each tag-encoding DNA shown in Table 1 can be replaced in favor of construction and expression of recombinant proteins, which assemble the regularly-weighted protein molecular weight markers;
        (3) in order to add shorter artificial peptide sequences and adjust the molecular weight of A in formula (I), or to add new restriction enzyme cutting sites, one or more bases of each tag-encoding DNA shown in Table 1 can be added and/or, eliminated in favor of construction and expression of recombinant proteins, which assemble the regularly-weighted protein molecular weight markers;
        (4) in order to enhance the recognition sensitivity of anti-tag antibodies, base(s) of each tag-encoding DNA shown in Table 1 can be added and/or eliminated to create amino acid(s) helpful to recognition sensitivity of the anti-tag antibodies.
    
    


In a particular embodiment, the arrangement of tags assembling A is His-HA-T7-E-VSVg-V5-AU5-S-HSV-FLAG-Lumio-Glu-Glu-cMyc-AU1 and the molecular weight thereof is about 15.0 kDa.
In a preferred embodiment, the A of formula (I) is the polypeptide of SEQ ID NO: 1 and the molecular weight thereof is about 15.0 kDa.
B and C of formula (I) are independently any mutually identical or different polypeptides with the value of molecular weight being a multiple of 5, and m and n are independently 0 or any integer larger than 0. In some embodiments, B and C are electric neutral, hydrophilic and/or polypeptides relatively insusceptible to glycosylation to prevent inaccuracy problem of the present recombinant protein as protein molecular weight marker.
In a preferred embodiment, the peptide sequences of B and C are selected from the group consisting of amino acid sequence of thioredoxin comprising one or a plurality of SEQ ID NO: 2, amino acid sequence of glutathione-S-transferase comprising one or a plurality of SEQ ID NO: 3, amino acid sequence of maltose-binding protein comprising one or a plurality of SEQ ID NO: 4, and amino acid sequence comprising one or a plurality of SEQ ID NO: 5. Among these sequences, the arrangement of the amino acid sequence of thioredoxin comprising one or a plurality of SEQ ID NO: 2, the amino acid sequence of glutathione-S-transferase comprising one or a plurality of SEQ ID NO: 3, the amino acid sequence of maltose-binding protein comprising one or a plurality of SEQ ID NO: 4, and the artificial amino acid sequence comprising one or a plurality of SEQ ID NO: 5 is any possible permutation and combination. The flanking region of selected amino acid sequences mentioned previously may include the residual amino acid sequence resulted from recombinant protein construction. Those residual fragments may derive from partial commercially available vector sequences or restriction enzyme recognition sequences, designed for simplifying the recombinant protein construction.
For (B)m-A-(C)n of the present invention, the flanking region of (B)m, A, and (C)n may include the residual amino acid sequence resulted from recombinant protein construction. Those residual fragments may derive from partial commercially available vector sequences or restriction enzyme recognition sequences, designed for simplifying the recombinant protein construction.
For the recombinant protein of formula (I), if m=0 and n=0, it is indicated that the recombinant protein consists of polypeptide A; if m=0 and n>0, it is indicated that the N terminal of the recombinant protein is polypeptide A; if m>0 and n=0, it is indicated that the C terminal of recombinant protein is polypeptide A; if m>0 and n>0, it is indicated that there are other amino acid sequences at the two ends of polypeptide A. In a preferred embodiment, m is 0 and n is 0 or larger than 0, such as various recombinant proteins shown in FIG. 1. The 15.0 kDa recombinant protein is a polypeptide of m=0, n=0, and A is SEQ ID NO: 1; the 20.0 kDa recombinant protein is the polypeptide of m=0, n=1, A is SEQ ID NO: 1, and C is an artificial amino acid sequence of SEQ ID NO: 5.
The auto-developing and regularly-weighted protein molecular weight marker kit of the present invention further comprises a solvent for stabilizing recombinant proteins. The solvent for stabilizing recombinant proteins extends the shelf-life of the recombinant protein and prevents the protease degradation. The solvent includes but not limits to Tris-H3PO4, ethylene diamine tetraacetic acid (EDTA), sodium dodecyl sulfate (SDS), dithiothreitol (DTT), NaN3 and/or glycerol with proper concentration and/or pH value.
According to the requirement, a plurality of recombinant proteins having formula (I) is selected for the protein molecular weight marker kit of the present invention. In some embodiments, the plurality of recombinant proteins having formula (I) are selected from the group consisting of a polypeptide of SEQ ID NO: 1 (M.W. is about 15.0 kDa), a polypeptide of SEQ ID NO: 6 (M.W. is about 20.0 kDa), a polypeptide of SEQ ID NO: 7 (M.W. is about 25.0 kDa), a polypeptide of SEQ ID NO: 8 (M.W. is about 30.0 kDa), a polypeptide of SEQ ID NO: 9 (M.W. is about 40.0 kDa), a polypeptide of SEQ ID NO: 10 (M.W. is about 50.0 kDa), a polypeptide of SEQ ID NO: 11 (M.W. is about 60.0 kDa), a polypeptide of SEQ ID NO: 12 (M.W. is about 80.0 kDa), a polypeptide of SEQ ID NO: 13 (M.W. is about 100.0 kDa), a polypeptide of SEQ ID NO: 14 (M.W. is about 120.0 kDa), a polypeptide of SEQ ID NO: 15 (M.W. is about 160.0 kDa), a polypeptide of SEQ ID NO: 16 (M.W. is about 180.0 kDa), a polypeptide of SEQ ID NO: 17 (M.W. is about 200.0 kDa), and a polypeptide of SEQ ID NO: 18 (M.W. is about 240.0 kDa), which are shown in FIG. 1.
For example, if there is a need for preparing protein molecular weight marker kit suitable for low molecular weight, SEQ ID NO. 1 (MW 15.0 kDa), SEQ ID NO. 6 (MW 20.0 kDa), SEQ ID NO. 7 (MW 25.0 kDa), SEQ ID NO. 8 (MW 30.0 kDa), SEQ ID NO. 9 (MW 40.0 kDa), SEQ ID NO. 10 (MW 50.0 kDa), SEQ ID NO. 11 (MW 60.0 kDa), SEQ ID NO. 12 (MW 80.0 kDa), SEQ ID NO. 13 (MW 100.0 kDa), and SEQ ID NO. 14 (MW 120.0 kDa) are selected to obtain the protein molecular weight marker kit used for low molecular weight.
Based on the well-known protein electrophoresis technology, the auto-developing and regularly-weighted protein molecular weight marker kit is used as protein marker for SDS-PAGE. For each and every recombinant protein of the present protein molecular weight marker kit contains the amino acid sequences of one or more commercial tags, users applying the present protein molecular weight marker kit in Western blotting do not need to buy new antibodies if the target protein contains the same protein marker(s). It is because that all the recombinant proteins has the amino acid sequence of SEQ ID NO: 1 that can be recognized by antibody selected from the group consisting of anti-His6-tag antibody, anti-HA-tag antibody, anti-T7-tag antibody, anti-E-tag antibody, anti-VSV-g-tag antibody, anti-V5-tag antibody, anti-AU5-tag antibody, anti-5-tag antibody, anti-HSV-tag antibody, anti-FLAG-tag antibody, anti-Lumio-tag antibody, anti-Glu-Glu-tag antibody, anti-cMyc-tag antibody, and anti-AU1-tag antibody. At the same time, the present protein molecular weight marker kit is also useful as positive control.
The present invention also provides a method for preparing the auto-developing and regularly-weighted protein molecular weight marker kit comprising
(a) constructing vectors, which comprise a plurality of nucleotide sequences encoding recombinant proteins of formula (I) independently, to obtain recombinant protein expression vectors,

(B)m-A-(C)n  (I),

wherein A is a polypeptide of SEQ ID NO: 1, B and C are independently any mutually identical or different polypeptides with the value of molecular weight being a multiple of 5, and m and n is independently 0 or any integer larger than 0;
(b) transforming the recombinant protein expression vectors into competent cells;
(c) selecting the competent cells carrying the recombinant protein expression vectors;
(d) inducing the competent cells carrying the recombinant protein expression vectors to express the recombinant proteins by administrating a inducer, such as IPTG; and
(e) extracting each of recombinant protein with different molecular weights independently, and mixing a plurality of recombinant proteins with one or more recombinant protein stabilizing solvents.
For increasing protein purity, protein purification is further performed by using various well-known protein purification technologies, such as affinity column and S200 gel filtration, before above-mentioned step (e).
In some embodiments, the above-mentioned plurality of nucleotide sequences encoding recombinant protein of formula (I) are selected from the group consisting of a nucleotide sequence encoding the polypeptide of SEQ ID NO: 1, a nucleotide sequence encoding the polypeptide of SEQ ID NO: 6, a nucleotide sequence encoding the polypeptide of SEQ ID NO: 7, a nucleotide sequence encoding the polypeptide of SEQ ID NO: 8, a nucleotide sequence encoding the polypeptide of SEQ ID NO: 9, a nucleotide sequence encoding the polypeptide of SEQ ID NO: 10, a nucleotide sequence encoding the polypeptide of SEQ ID NO: 11, a nucleotide sequence encoding the polypeptide of SEQ ID NO: 12, a nucleotide sequence encoding the polypeptide of SEQ ID NO: 13, a nucleotide sequence encoding the polypeptide of SEQ ID NO: 14, a nucleotide sequence encoding the polypeptide of SEQ ID NO: 15, a nucleotide sequence encoding the polypeptide of SEQ ID NO: 16, a nucleotide sequence encoding the polypeptide of SEQ ID NO: 17, and a nucleotide sequence encoding the polypeptide of SEQ ID NO: 18.
The next examples provide some exemplary embodiments of the present invention as follows:
EXAMPLES
The examples below are non-limiting and are merely representative of various aspects and features of the present invention.
Example 1
FIG. 1 is a schematic diagram of protein markers containing 14 tags at N-terminal and having different molecular size. 14 tags of most common-used in current proteomic studies (shown in Table 1) were selected and used as “locomotive” of the present protein marker. Other proteins were sequentially connected to the 14 tags to form ladder-like protein markers with regular molecular weights. During color development operating procedure of Western blotting, if antibodies recognizing any one of the 14 tags were used, and the HRP-conjugated secondary antibodies were then employed to recognize the anti-tag antibodies, ladder-like protein markers with regular molecular weight can be auto-developed on the film; convenience and accuracy of protein-related studies were therefore increased. 14-tag was used as a beginning in this example, and proteins with different molecular weights, TRX (thioredoxin, molecular weight is about 10.0 kDa), GST (glutathione-S-transferase, molecular weight is about 25.0 kDa), or MBP (maltose-binding protein, molecular weight is about 40.0 kDa), were connected sequentially to construct protein marker with specific size.
Example 2
Construction of Genes Encoding 14-Tag, GST, MBP, TRX
The required primers listed in Table 2 were ordered.







TABLE 2







primers used for annealing 14-tag by assembly-PCR










Length




(Repeating



Primer No.
Sequence Length)
Sequence 5′ → 3′





P1
57
SEQ ID NO: 46




GGAACGCCATATGCACCATCATCATCATCATTATCCTT




ACGATGTTCCAGATTATGC


 


P1new
40
SEQ ID NO: 47




GGAATTCCATATGCGGGGTTCTCATCATCATCATCATC




AT


 


P2-A
57 (18)
SEQ ID NO: 48




CCACCAGTCATACTGGCCATGATGTAACGGTAGGTGT




CAGCATAATCTGGAACATCG


 


P3
57 (18)
SEQ ID NO: 49




GGCCAGTATGACTGGTGGACAGCAAATGGGTGCGCC




GGTGCCGTATCCGGACCCACT


 


P4-A
57 (18)
SEQ ID NO: 50




CCCAACCGGTTCATCTCGATGTCGGTGTAACGCGGTT




CCAGTGGGTCCGGATACGGC


 


P5
57 (18)
SEQ ID NO: 51




CGAGATGAACCGGTTGGGCAAGCCTATCCCTAACCCT




CTCCTCGGTCTCGATTCTAC


 


P6-A
57 (18)
SEQ ID NO: 52




TCAAATTTAGCAGCAGCGGTTTCCTTCAGGTAGAAGT




CCGTAGAATCGAGACCGAGG


 


P7
57 (18)
SEQ ID NO: 53




CGCTGCTGCTAAATTTGAGCGCCAGCACATGGACAGC




CAGCCAGAACTCGCTCCTGA


 


P8-A
57 (18)
SEQ ID NO: 54




CAACACTTATCGTCGTCATCCTTGTAATCCTCTGGGTC




TTCAGGAGCGAGTTCTGGC


 


P9
57 (18)
SEQ ID NO: 55




TGACGACGATAAGTGTTGTCCTGGCTGTTGCGAAGAA




GAAGAATACATGCCGATGGA


 


P10-A
58 (18)
SEQ ID NO: 56




CTAGCTAGCTCACAGATCCTCTTCTGAGATGAGTTTTT




GTTCCATCGGCATGTATTCT




TCAGAAGAGGATCTGCTCGAGGGTGATGTTAAACTTA




CCCAATCGTACGCTAGCTAG




Reverse primer (SEQ ID NO: 57):




AGAATACATGCCGATGGAACAAAAACTCATCTCAGAA




GAGGATCTGTGAGCTAGCTAG


 


P11-A
57 (15)
SEQ ID NO: 58




CTAGCTAGCGTACGATTGGGTAAGTTTAACATCACCCT




CGAGCAGATCCTCTTCTGA




Reverse primer (SEQ ID NO: 59):




TCAGAAGAGGATCTGCTCGAGGGTGATGTTAAACTTA




CCCAATCGTACGCTAGCTAG









The sequences of the initial and the end primers (P1 and P10-A) contained the designed restriction enzyme cutting sites. Assembly-PCR was utilized to anneal these primers into one fragment, and the fragment was inserted to pBlunt vector (Invitrogen). The obtained recombinant plasmid was named pBlunt-15 kDa. To conduct PCR, P1new and P10-A were then used as primers and pBlunt-15 kDa was used as template, and the PCR fragments were ligated to pBlunt vector to obtain pBlunt-RGS-15 kDa plasmid. Subsequently, P1new and P11-A (without stop codon) were used as primers and pBlunt-15 kDa was used as template for conducting PCR, and the PCR fragments were ligated to pBlunt vector. The obtained product was named pBlunt-20 kDa plasmid.
In addition, the primers that were designed to contain special cutting sites were as Table 3.







TABLE 3







Primers for PCR cloning of TRX, MBP and GST










Length




(Repeating



Primer No.
Sequence Length)
Sequence 5′ → 3′





GST-P1
24 (18)
SEQ ID NO: 60




CTCGAGATGTCCCCTATACTAGGT


 


GST-P2-A
59 (14)
SEQ ID NO: 61




GCTAGCTCAGAATTCGTCGACGATATCGTTAACCCAT




GGATGCATATACTTGCTGGATT


 


MBP1-P1
58 (18)
SEQ ID NO: 62




GTCGACATGCATAAAATCGAAGAAGGTAAACTGGTAA




TCTGGATTAACGGCGATAAAG


 


MBP1-P2-A
58 (18)
SEQ ID NO: 63




GTTAACGGCTTCATCGACAGTCTGACGACCGCTGGCG




GCGTTGATCACCGCAGTACGC


 


MBP2-P1
58 (18)
SEQ ID NO: 64




GTTAACAAAATCGAAGAAGGTAAACTGGTAATCTGGA




TTAACGGCGATAAAGGCTAT


 


MBP2-P2-A
58 (18)
SEQ ID NO: 65




CTGCAGGTCGACGATATCCTAAAGCTTCCATGGGATAT




CGGCTTCATCGACAGTCTGA


 


TRX-P1
52 (18)
SEQ ID NO: 66




GTCGACGAATTCAGCGATAAAATTATTCACCTGACTG




ACGACAGTTTTGACA


 


TRX-P2-A
50 (18)
SEQ ID NO: 67




GCTAGCAAGCTTACCCACTTTGGTTGCCGCCACTTCA




CCGTTTTTGAACA









PCR technique was utilized to insert gene fragments, such as GST, MBP, and TRX, into pBlunt vectors (pBlunt-GST′pBlunt-MBP′pBlunt-TRX). The plasmids were transformed into TOP10 E. coli to select a colony. Plasmid DNA was extracted from selected colony and cut by restriction enzyme to confirm the size of sequence. The colony containing fragment of correct size was subjected to further sequencing analysis.
1. Assembly-Polymerase Chain Reaction
PCR Reaction Solution:














10× PCR buffer (Taq DNA polymerase buffer)
5
μl


2.5 mM dNTP
5
μl


primer mixture
1
μl


ddH2O
37.3
μl


Cell cDNA
1
μl


Taq DNA polymerase
0.5
μl


Pfu DNA polymerase
0.2
μl


Total Volume
50
μl/reaction









Reaction Cycle:

94° C. 2 min→[94° C. 30 sec→55° C. 30 sec→72° C. 1 min]×30 circles→72° C. 5 min→4° C.

2. DNA Ligation Reaction Solution
















fragment to be inserted
  7 μl



vector (100~200 ng)
  1 μl



10× polymerase buffer
1.5 μl



ATP (10 mM)
1.5 μl



T4 DNA polymerase (U/μl)
1.5 μl



ddH2O
2.5 μl



Total Volume
 15 μl







Overnight reaction under 16° C. (16-24 hr)






3. Mini-Preparation of Plasmid DNA
One white colony was picked from the plate, and seeded into 3 ml LB medium (containing 1 μg/ml Ampicillin). After cultivation in incubator under 37° C. and 180 rpm for 14-16 hr, 1.5 ml of bacteria liquid was transferred to a 1.5 ml microcentrifuge tube and subjected to 6000 rpm centrifugation for 5 min under room temperature. The supernatant was removed; 150 μl of Solution I (50 mM Tris/HCl, 10 mM EDTA (pH 8.0), and 100 μg/ml ribonuclease) was added; and vortex to resuspend the pellet. 150 μl of Solution II (200 mM NaOH and 1% SDS) was added; and the tube was gently inverted for 6-8 times followed by stood still under room temperature for 3 min. 180 μl of 3M KOAc (pH 5.5) was added. After being gently inverted for 6 to 8 times (vortex was prohibited), the tube was rested under room temperature for 3 min followed by 12000 rpm centrifugation for 3 min. The supernatant (approximate 400 μl) was transferred to another new 1.5 ml microcentrifuge tube, added with equal volume of isopropanol (400 μl), and mixed thoroughly. After placing in −20° C. refrigerator, the tube was subjected to 12000 rpm centrifugation or 3 min. The supernatant was removed. 1 ml of 75% iced alcohol was added to wash the pellet, and the tube was subject to 12000 rpm centrifugation for 3 min. After removing of the supernatant, the alcohol was discarded and the pellet was air-dried in the vacuum oven. The pellet was resuspended with 20 μl ddH2O (the volume of ddH2O can be adjusted according to the size of pellet).
Example 3
Gene Construction of Recombinant Protein Markers


    (1) pRSETB-14tags (15 kDa) plasmids and pRSETB-14tags (20 kDa) plasmids were obtained by NdeI and HindIII digestion of pBlunt-15 kDa plasmid and pBlunt-20 kDa plasmid, which followed by ligation into pRSETB expression vector (Invitrogen).
    (2) TRX was inserted after 20 kDa with EcoRI and HindIII to obtain pRSETB-14tags-TRX (30 kDa) plasmid.
    (3) To obtain pRSETB-14tags-GST-stop-TRX (40 kDa) plasmid, an in-between sequence of 30 kDa recombinant protein was replaced with GST, which followed by a stop codon, by using XhoI and NheI.
    (4) To remove the stop codon between GST and TRX and thus obtain pRSETB-14tags-GST-TRX (50 kDa) plasmid, the pRSETB-14tags-GST-stop-TRX (40 kDa) plasmid was digested by EcoRI and then self-ligated.
    (5) To obtain pRSETB-14tags-TRX (25 kDa) plasmid, the in-between GST of the 50 kDa plasmid was removed by using XhoI and SalI; and the cutting sites of XhoI and SalI were ligated to each other to make the original restriction cutting site disappeared.
    (6) TRX was inserted into the sequence of 40 kDa plasmid by using HpaI and NheI to replace the stop codon after GST and form a pRSETB-14tags-GST-TRX-TRX (60 kDa) plasmid.
    (7) MBP, which was followed by a stop codon, was inserted into the 60 kDa plasmid by using HpaI and SalI; and a pRSETB-14tags-GST-MBP-stop-TRX-TRX (80 kDa) plasmid was thus formed.
    (8) For obtaining pRSETB-14tags-GST-MBP-TRX-TRX (100 kDa) plasmid, the 80 kDa plasmid was digested by EcoRV and self-ligated to remove the stop codon between MBP and TRX.
    (9) MBP-MBP was inserted into 40 kDa plasmid before the stop codon by using NsiI and HpaI; pRSETB-14tags-GST-MBP-MBP-stop-TRX (120 kDa) plasmid was then obtained.
    (10) For obtaining pRSETB-14tags-GST-MBP-MBP-MBP (160 kDa) plasmid, MBP-MBP was substituted for TRX-TRX of 100 kDa plasmid by using SalI and HindIII to make the plasmid carry continuous three MBPs.
    (11) For obtaining pRSETB-14tags-GST-MBP-MBP-MBP-TRX-TRX (180 kDa) plasmid, MBP-MBP was inserted into 100 kDa plasmid by using NsiI and HpaI to make the plasmid carry continuous three MBPs and two TRXs.
    (12) For obtaining pRSETB-14tags-GST-MBP-MBP-MBP-MBP (200 kDa) plasmid, MBP-MBP was substituted for the stop-TRX of 120 kDa plasmid by using SalI and HindIII to make the plasmid carry continuous four MBP.
    (13) For obtaining pRSETB-14tags-GST-MBP-MBP-MBP-MBP-MBP (240 kDa) plasmid, MBP-MBP was substituted for the last TRX-TRX of 180 kDa plasmid by using SalI and HindIII to make the plasmid carry continuous five MBP.


Example 4
E. coli Transformation
The competent cells strains, Top 10 and BL21 (DE3), prepared by our laboratory, were taken out from −80° C. refrigerator and unfrozen. 100 μl of culture was transferred to a 1.5 ml microcentrifuge tube, and 7.5 μl DNA annealing product or 1 μl DNA plasmid of protein marker was added into the tube and mixed with the culture thoroughly. After setting on ice for 30 min, the tube was placed in water bath to heat shock for 1 min and 30 sec under 42° C., and then it was placed on ice for 5 min. 600 μl LB (Luria-Broth) was added into the tube at a Laminar flow hood. The tube was incubated in 37° C. incubator for 45 min with vertical shake at 180-200 rpm, and then subjected to centrifugation at 5000 rpm for 5 min. About 550 μl of supernatant was discarded, and the cells were resuspended with about 150 μl of remaining medium. Finally, the bacteria solution was plated in LB plates containing Kanamycin (1 μg/ml, used to screen cells containing pBlunt-derived vectors) or Ampicillin (1 μg/ml, used to screen cells containing pRSETB-derived vectors), which were incubated in 37° C. incubator for 14 to 16 hr. Next day, a single white colony was picked from the plate and subjected to culture and amplification.
Example 5
Large Induction of Recombinant Protein Marker Expression
A successful transformed colony was picked from the plate and seeded in 3 ml Ampicillin-containing LB (1 μg/ml). After cultivating in 37° C. incubator for several hours, the culture was poured into a flask with 500 ml of Ampicillin-containing LB medium (1 μg/ml) and cultivated in 37° C. incubator until the OD600 reached 0.2-0.3. 1M isopropyl β-D-1-thiogalactopyranoside (IPTG) was added into the culture to the final concentration of 0.5 mM. The culture was cultivated in 25° C. incubator until the OD600 reached 0.6-0.8. The bacteria were pelleted by supercentrifugation at 6000 rpm, and the pellet was stored frozen after removing the supernatant.
Example 6
Monitoring the Expression of Recombinant Protein
1. SDS-PAGE Gels Preparation
10% or 12.5% SDS-PAGE running gel solution was prepared and pipetted into glass electrophoresis cell carefully. A small amount of distilled water was added carefully with dropper to produce a horizontal surface of the running gel during condensation. The gel was set for 30 min until solidified. In the following, 3% stacking gel was prepared. After discarding distilled water, the stacking gel was pipetted onto the running gel and comb with appropriate thickness was inserted to create wells for loading samples. The gel was set for another 30 min until the stacking gel solidified.
2. Analysis of SDS-PAGE Gel Electrophoresis
The bacteria harvested previously were resuspended with 50 μl PBS buffer (50 μl PBS buffer for per ml of bacteria solution pellet). 80 μl bacteria and 20 μl 6× reducing buffer were mixed thoroughly, heated at 95° C. for 5 min to make protein denature, and then placed on ice. 10-20 μl of sample was loaded in each well of upper-layer stacking gel. Electrophoresis in stacking gel was performed with electric current of 25 mA, and the following electrophoresis in running gel was performed under 158V. After finishing of the electrophoresis, the Western blotting was applied for analysis.
3. Western Blotting
SDS-PAGE was utilized to separate the protein. After that, SDS-PAGE was removed and rinsed in elector-transfer buffer to transfer the proteins from SDS-PAGE to nitrocellulose membrane (under voltage of 94V for about 1.5 hr). The nitrocellulose membrane was blocked with blocking buffer (PBS containing 5% fat-free milk) at 4° C. overnight and washed once by PBST (i.e. PBS+Tween surfactants) for 15 min in the next day. The needed primary antibody (one of the anti-tags-antibodies, listed in Table 4) was diluted with blocking buffer. The nitrocellulose membrane was reacted with blocking buffer-diluted antibody, accompanied constant shaking, at room temperature for 1 hr.







TABLE 4







the brand and dilution ratio of antibodies used in the Western blot












Brand Name
Dilution


Antibody Name
Species
(Catalog no.)
ratio





AU1
Goat
Bethyl(A190-124A)
1/2000


cMyc
Mouse
Self production (ascites)
1/7500


E-tag
Goat
Bethyl(A190-132A)
1/2000


Flag
Mouse
Sigma
1/7500


GluGlu
Goat
Bethyl(A190-110A)
1/2000


HA
Mouse
Covance
1/4000


His
Mouse
Serotech
1/200


HSV
Goat
Bethyl(A190-136A)
1/2000


S-tag
Goat
Bethyl(A190-134A)
1/4000


T7
Goat
Bethyl(A190-116A)
1/2000


V5
Mouse
Sigma(V8012)
1/4000


VSV-G
Goat
Bethyl(A190-130A)
1/2000


Anti-goat IgG-HRP
Rabbit
Jackson
1/2000


Anti-mouse IgG-Fc-HRP
Goat
Jackson
1/2000









After repeating the 15 min washing step three times, the nitrocellulose membrane was reacted with HRP-conjugated antibody, which was diluted 2000 times with blocking buffer, on shaker at room temperature for 1 hr. The 15 min washing step was repeated three times. ECL reagents (Amersham) were utilized to detect conjugated antibodies. Equal volumes of reagent A and reagent B were mixed, and the nitrocellulose membrane was soaked in the mixture for 2-3 min. After that, the excess reagent was removed by towels. The nitrocellulose membrane was placed into cassette, and then an X-ray film was placed on the nitrocellulose membrane. The film was exposed for 3-60 sec and subjected to development. All the ECL procedures were operated in darkroom or detect the image by chemiluminescent detection system (Bio-rad) of Department of Biotechnology, Kaohsiung Medical University.
Results
1. Screening the Basic Recognition Unit and Increasing Molecular Weight Unit
The subject of the present invention is to develop an auto-developing protein marker. First of all, the commonly used tags were selected and utilized as recognition unit of protein marker. By searching information about frequently used tags from current catalogs and references, the most commonly used tags and the sequences thereof were generalized (listed in previous Table 1) and used as the main framework of recombinant protein markers. These tags were all popularly used by current researchers. The protein marker would auto-develop only if researchers applied appropriate anti-tag antibodies, which dramatically increased user's convenience.
After identifying proper tags as the basic recognition unit of recombinant protein marker, other protein sequences were selected for assembling of protein markers with different molecular weights. Through a series of search, GST, MBP, and TRX were selected and used as fusing proteins that connected with the basic recognition unit. In addition to the benefit of increasing water solubility, these three proteins could be used as tags for purifying and antibody recognition. The 14-tag basic recognition unit was combined with these three proteins with various repetitions to create protein markers of different molecular weights (FIG. 1). Repetition of restriction enzyme cutting sites during assembly procedure can be prevented by designing their locations and order in advance (FIG. 2).
2. Gene Cloning of 14-Tag Basic Recognition Unit of Recombinant Protein Markers
The selected 14 sequences of commonly used tags were constructed into a recombinant 14-tag basic recognition unit, which was served as the basic component of protein markers with different molecular weights. The 10 primers containing sequences of these 14 tags were designed for assembly PCR to anneal primers into a 15 kDa fragment (FIG. 3A). Analyzing by gel electrophoresis, the size of assembly PCR product, as expected, was 409 bp (FIG. 3B). After that, the products were inserted into pBlunt vector and the insertion order was checked by NdeI and NheI. In order to further confirm, the plasmid was sequenced to check its sequence. On the other hand, P11-A primer was substituted for the tenth primer to anneal another 408 bp fragment, and a basic recognition unit (20 kDa) with different molecular weight, which could be used for assembling protein markers with different molecular weights, was thus obtained by the same method. To form two basic recognition units of 14-tag, restriction enzymes, NdeI and HindIII, were used to insert 15 kDa and 20 kDa sequence into pRSETB expression vector respectively.
3. Gene Cloning of GST, TRX and MBP
The primers comprising the designed restriction enzyme cutting sites was used to amplify the plasmids containing GST, TRX or MBP gene by polymerase chain reaction (PCR). Those amplified fragments were annealed into pBlunt vector and then cut by restriction enzymes. Gel electrophoresis was used to prove that the size of cutting fragments were as expected: MBP was 1134 bp, GST was 645 bp, and TRX was 300 bp (FIG. 3 C-D). Accuracy of the sequences was further confirmed by sequencing process. These three proteins were used as constructing material for increasing molecular weight of recombinant protein marker. The size of MBP protein was about 39.6 kDa; GST protein was about 25 kDa; and TRX protein was about 10 kDa.
4. Gene Cloning of the Recombinant Proteins Having Different Molecular Weight
When construction of the main structure of 14-tag, GST, TRX and MBP was completed, Western blotting was utilized to confirm the basic recognition unit was able to be recognized by different anti-tag antibodies (FIG. 4). The protein markers were then assembled based on the designed restriction enzyme map (FIG. 2). Since the main structure and material for construction of plasmid DNA has been sequenced, another sequencing procedure was omitted after the subcloning but restriction enzymes digestion and agarose gel electrophoresis were done to confirm that the inserted fragments were consistent with the expectation (FIG. 5 A-F and FIG. 6 A-F).
5. Ensuring the Expression of Recombinant Protein Markers
After constructing the plasmids encoding the protein markers with different molecular weights, these plasmids were transformed into BL21 (DE 3) E. coli, which was served as protein expression system. The transformed E. coli was cultured for a short time, and 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was then added to the cultures for inducing protein expression. The result of SDS-PAGE analysis (FIG. 7) confirmed that the transformed recombinant protein markers were expressed by E. coli. Various antibodies (shown in previous Table 4) were used to perform Western blotting, and the results confirmed the ability of E. coli to express recombinant protein markers with correct size, which could be recognized by various antibodies and shown as products with same molecular weight. The outcomes showed that all the protein markers having different molecular weights comprised the 14-tag basic recognition unit (FIG. 8 A-C).
6. Making the Combination of Recombinant Protein Markers and Analyzing its Commercialization Potential
The plasmid DNAs having different molecular weights were transformed into E. coli and recombinant proteins expression thereof was induced respectively. Bacteria were subjected to centrifugation followed by formulated into sample solution with reducing dye. The protein markers with different molecular weights were mixed in different ratio. After adjusting ratio and volume for several times, the combination was completed. At last, Western blotting was used to prove that the combination of recombinant protein markers with different molecular weights was able to be recognized by various anti-tag antibodies and represented ladder-like bands clearly on the film (FIG. 9 A-D and FIG. 10 A-C). The results displayed that there were background noises between 80 kDa and 100 kDa, and also between 30 kDa and 40 kDa. The strength of background noise was altered while adding different antibodies, suggesting that the background noise may result from non-specific binding of the antibodies.
The result of Western blotting (FIG. 9 D) was further analyzed to obtain the Rf (retention factor) value. It could be found that the molecular weight of the present recombination protein markers was positively correlated to the migration distance of the proteins. The log value of molecular weight and migration distance of each recombinant protein marker of present invention was shown in Table 5.







TABLE 5







The log value of molecular weight (log M.W.) and the migration distance


of each recombinant protein markers of present invention









M.W. (kDa)
Log M.W.
Migration Distance (mm)












100
2.00
4


80
1.90
6


60
1.78
8.5


50
1.70
10.5


40
1.60
13


30
1.48
16.5


25
1.40
18


20
1.30
22


15
1.18
28.5









The linear regression of log value of molecular weight versus migration distance was performed (FIG. 11 A-B). The results demonstrated that the coefficient determination (R2) of commercial protein marker (FERMANTAS) was 0.9821, whereas the R2 of low range protein markers of the present invention was 0.9723 (FIG. 11 B), which is lower than that of the commercial protein marker.
One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The animals, processes and methods for producing them are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.

## Claims

1. An auto-developing and regularly-weighted protein molecular weight marker kit, which comprises:
(a) a plurality of recombinant proteins having formula (I),


(B)m-A-(C)n  (I),


wherein A is the polypeptide of SEQ ID NO: 1, B and C are independently any mutually identical or different polypeptides with the value of molecular weight being a multiple of 5 kDa, and m and n are independently 0 or any integer larger than 0; and
(b) one or more solvents for stabilizing the recombinant proteins.

2. The auto-developing and regularly-weighted protein molecular weight marker kit of claim 1, wherein B and C are selected from thioredoxin comprising the amino acid sequence of SEQ ID NO: 2, glutathione-S-transferase comprising the amino acid sequence of SEQ ID NO: 3, maltose-binding protein comprising the amino acid sequence of SEQ ID NO: 4, or artificial peptide sequence comprising the amino acid sequence of SEQ ID NO: 5.

3. The auto-developing and regularly-weighted protein molecular weight marker kit of claim 1, wherein the plurality of recombinant proteins having formula (I) are selected from the group consisting of the polypeptide of SEQ ID NO: 1, the polypeptide of SEQ ID NO: 6, the polypeptide of SEQ ID NO: 7, the polypeptide of SEQ ID NO: 8, the polypeptide of SEQ ID NO: 9, the polypeptide of SEQ ID NO: 10, the polypeptide of SEQ ID NO: 11, the polypeptide of SEQ ID NO: 12, the polypeptide of SEQ ID NO: 13, the polypeptide of SEQ ID NO: 14, the polypeptide of SEQ ID NO: 15, the polypeptide of SEQ ID NO: 16, the polypeptide of SEQ ID NO: 17, and the polypeptide of SEQ ID NO: 18.

4. The auto-developing and regularly-weighted protein molecular weight marker kit of claim 1, wherein the polypeptide of SEQ ID NO: 1 is recognized by an antibody selected from the group consisting of anti-His6-tag antibody, anti-HA-tag antibody, anti-T7-tag antibody, anti-E-tag antibody, anti-VSV-g-tag antibody, anti-V5-tag antibody, anti-AU5-tag antibody, anti-S-tag antibody, anti-HSV-tag antibody, anti-FLAG-tag antibody, anti-Lumio-tag antibody, anti-Glu-Glu-tag antibody, anti-cMyc-tag antibody, and anti-AU1-tag antibody.

5. The auto-developing and regularly-weighted protein molecular weight marker kit of claim 1, which is used as protein marker for SDS-PAGE.

6. The auto-developing and regularly-weighted protein molecular weight marker kit of claim 1, which is used as protein marker and/or positive control of Western blotting utilized antibodies below for color development: anti-His6-tag antibody, anti-HA-tag antibody, anti-T7-tag antibody, anti-E-tag antibody, anti-VSV-g-tag antibody, anti-V5-tag antibody, anti-AU5-tag antibody, anti-S-tag antibody, anti-HS V-tag antibody, anti-FLAG-tag antibody, anti-Lumio-tag antibody, anti-Glu-Glu-tag antibody, anti-cMyc-tag antibody, or anti-AU1-tag antibody.

7. A method for preparing an auto-developing and regularly-weighted protein molecular weight marker kit comprising:
(a) constructing vectors, which comprises a plurality of nucleotide sequences encoding recombinant proteins of formula (I) independently, to obtain recombinant protein expression vectors,


(B)m-A-(C)n  (I),

wherein A is the polypeptide of SEQ ID NO: 1, B and C are independently any mutually identical or different polypeptides with the value of molecular weight being a multiple of 5 kDa, and m and n are independently 0 or any integer larger than 0;

(b) transforming the recombinant protein expression vectors into competent cells;
(c) selecting the competent cells carrying the recombinant protein expression vectors;
(d) inducing the competent cells carrying the recombinant protein expression vectors to express the recombinant proteins by administrating an inducer; and
extracting each of recombinant protein with different molecular weights independently, and mixing a plurality of recombinant proteins with one or more recombinant protein stabilizing solvents.

8. The method of claim 7, which further comprises a step of purifying the recombinant proteins, which is performed by using an affinity column or 5200 gel filtration, before mixing the plurality of recombinant proteins with one or more recombinant protein stabilizing solvents to increase the recombinant protein purity.

9. The method of claim 7, wherein the plurality of nucleotide sequences encoding recombinant protein of formula (I) are selected from the group consisting of a nucleotide sequence encoding the polypeptide of SEQ ID NO: 1, a nucleotide sequence encoding the polypeptide of SEQ ID NO: 6, a nucleotide sequence encoding the polypeptide of SEQ ID NO: 7, a nucleotide sequence encoding the polypeptide of SEQ ID NO: 8, a nucleotide sequence encoding the polypeptide of SEQ ID NO: 9, a nucleotide sequence encoding the polypeptide of SEQ ID NO: 10, a nucleotide sequence encoding the polypeptide of SEQ ID NO: 11, a nucleotide sequence encoding the polypeptide of SEQ ID NO: 12, a nucleotide sequence encoding the polypeptide of SEQ ID NO: 13, a nucleotide sequence encoding the polypeptide of SEQ ID NO: 14, a nucleotide sequence encoding the polypeptide of SEQ ID NO: 15, a nucleotide sequence encoding the polypeptide of SEQ ID NO: 16, a nucleotide sequence encoding the polypeptide of SEQ ID NO: 17, and a nucleotide sequence encoding the polypeptide of SEQ ID NO: 18.