CN105063074B - A kind of method of artificial reconstructed functional protein - Google Patents
A kind of method of artificial reconstructed functional protein Download PDFInfo
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Abstract
The invention discloses a kind of methods of artificial reconstructed functional protein, based on target protein molecular structure, choose library construction recombination module, the module is secondary building unit (alpha-helix, beta-pleated sheet, β-corner), and including important active site and ligand binding site region, by the way that the module region gene is introduced Linker, and digestion is carried out using restriction endonuclease, endonuclease bamhi completely random connection combination building library of recombinant, expresses and screens.The literature data of the method building is huge, function orientation is controllable, pass through the analysis to new functional protein structure, understand effective module recombination and effective module in new functional protein, improves design and rational protein to understand structure function biological information and the auxiliary of target protein and help is provided.
Description
Background technique:
The artificial reconstructed evolution of functional protein is that the structure for changing protein and property meet effective way of industrialization demand
Diameter, present directed evolution technologies have been used for the evolution of a enzymes up to a hundred, the activity and efficiency of biological enzyme are substantially increased, in work
Industry, agricultural, bio-pharmaceuticals etc. produce tremendous influence.
The success or not of the directed evolution of functional protein depend primarily on constructed mutated library whether have it is sufficiently high
Diversity and storage capacity.According to the difference of library construction principle, rationality can be divided into and evolved and two kinds of strategies of stochastic evolution.Rationality
Evolution strategy needs the structure of ex ante analysis protein, prediction and building three-dimensional structure.Rationality evolution is only applicable to structure and function
More clear protein, albumen indefinite for molecular structure are difficult to be transformed relationship between energy.Thus, at random into
Chemical conversion is highly effective Study on Directed Evolution of Proteins means.Reorganize skill using fallibility PCR as the random mutation technology of representative and with DNA
Art is that the gene recombination technology of representative is currently used two kinds of stochastic evolution technologies.Fallibility round pcr is deposited in practical applications
In certain limitation, it is by changing the Mg in PCR reaction system2+With the concentration of dNTPs, change the guarantor of archaeal dna polymerase
True property makes a variation so that successive or combined type is accumulative come the method for obtaining ideal abrupt body.What it is due to technology introducing is point mutation, this
Result in the speed for obtaining ideal abrupt body slower, and target gene length is too short (being less than 800bp).It is produced relative to fallibility PCR
Raw point mutation is that one kind more effectively establishes mutant library method based on the recombination between gene order.Currently, DNA weight
There are many method of group, such as DNA reorganization, staggeredly extension recombination, random primer vitro recombination.These methods are by by parental gene
Segment is mixed and connects to obtain ideal abrupt body.Mutation random combine between parent can not only be easier to by DNA shuffling technology
Mutant is obtained, it can also be by the superior set and one between parent.But homologous gene recombination cannot be used for homology and be lower than
The gene order of 70%-80%, therefore effective mutant obtained is few.
1998, Ostermeier et al. established gradual cutting and generates heterozygosis zymotechnic (ITCHY), had started non-same
The beginning of source property recombination, has been developed multinomial non-homogeneous recombinant technique at present, does not depend on the protein recombination of sequence homology such as
Method (SHIPREC) and non-homogeneous random recombination (NRR) etc..But these methods are by the structural domain of DNA and important function
Fragment collapses reduce the validity in constructed library.For the ratio for increasing functional individual, researcher, which develops, utilizes albumen
The method that matter structure function segment carries out recombination to construct gene library, such as Block Shuffling, Domain Shuffling and
Module Shuffling etc..But these method recombination module sequences are long and quantity is few, the library of recombinant multiplicity of formation
Property is low.2008, Graziano etc. was recombination module using Secondary structure unit, successfully obtained soluble protein.
Graziano etc. searches from PDB Protein Data Bank first and chooses the secondary structure of all proteins in Escherichia coli, choosing
The secondary structure types (α-helix, β-strand, loop-H, loop-S) of 4 kinds of protein are taken, while according to different two
Level structure designs 16 kinds of difference Linker, forms DNA double chain gene pool first with the method for PCR, is added in restriction nuclease
Enzyme cutting digests double-stranded DNA and forms cohesive end, and DNA fragmentation is connected into long-chain using ligase, upstream and downstream primer PCR is added and expands
It after increasing, connects in vector introduction recipient bacterium, is expressed and screened.This method need to design a plurality of Linker, design
Linker length reaches 54bp, and same secondary structure, which needs to design, connects different Linker, increases experimental cost；It is connected as
Semi-random connection reduces the storage capacity in library.
Summary of the invention:
The purpose of the present invention is overcoming defect and deficiency of the existing technology, a kind of functional protein orientation is provided for people
The method of evolution.
A kind of method of artificial reconstructed functional protein of the present invention chooses text based on target protein molecular structure
Library constructs recombination module, and the module is secondary building unit (alpha-helix, beta-pleated sheet, β-corner), and is urged including important
Change active site and ligand binding site region, by the way that the module region gene is introduced Linker, and uses restricted core
Sour restriction endonuclease carries out digestion, and endonuclease bamhi completely random connection combination building library of recombinant is expressed and screened.
A kind of method of artificial reconstructed functional protein, specific step are as follows:
(1) according to target enzyme protein structure, inquiry obtains the protein sequence in albumen database, chooses library structure
Recombination module is built, the module is its secondary building unit (alpha-helix, beta-pleated sheet, β-corner), and including important catalysis
Active site and ligand binding site region；
(2) genetic fragment of each module region is determined；
(3) by PCR or it is artificial synthesized in the way of at selected genetic fragment both ends Linker is added, form both ends
The single stranded DNA of Linker；
(4) using the target enzyme protein sequence head and the tail genetic fragment as termination sequence, and only in the termination sequence one
Linker is added in end, forms the single stranded DNA of one end Linker；
(5) under the action of polymerase, step 3 and step 4 gained single stranded DNA are synthesized into double-stranded DNA, use is restricted
Double-stranded DNA described in endonuclease digestion generates cohesive end, forms endonuclease bamhi；
(6) in the case where connecting enzyme effect, step 5 gained endonuclease bamhi is connected, forms library of recombinant；
(7) library of recombinant after connection is carried out PCR amplification by the amplification and expression of the gene library, and amplification is drawn
Object introduces restriction enzyme site, and recycling is not less than the DNA fragmentation of original gene length areas, connect after digestion with carrier, express and sieve
Choosing.
Main sections product length after step (6) connection is less than the target protein gene order length, passes through weight
Poly- PCR is extended；
The Linker connects the multiple that the base number of the gene order of two modules is 3, the ammonia after translation after digestion
Base acid is flexible amino acid.
Further, it is 9 bases that the Linker connects the length of the gene order of two modules after digestion, is reduced
The Linker ratio in sequence in the reassembled, reduces influence of the Linker to library function.
The method Linker forms the cohesive end of same complementation after digestion, therefore ensure that in library of recombinant
When be intermodule completely random combination.
This method is combination basic module using Secondary structure and important functional structure region, is contained with one
For having 20 modules, random combine can generate 2020The different combined sequence mode of kind, has huge combined sequence space,
The diversity in constructed library is improved, while using Secondary structure and important functional structure region as module, benefit
With original functional structure, the validity of combinatorial libraries is improved, promotes the directed evolution transformation of functional protein.
The objective function albumen is one or more protein, and a certain protein function directional transformation can also be used
The secondary structure of multiple proteins creates new protein.
The module is based on single secondary building unit, and the amino acid number contained is 3-50, preferred amino acid
Number is the module of 3-20, and module amino acid number is few, and it is more that target protein contains number of modules, constructed recombination library diversity
It is high.
The number of modules that the gene library that the module is formed after connection recombination is included ensure that library not less than 6
Diversity.
The cohesive end be non-palindromic sequence, guarantee connect recombination when double-stranded DNA will not Opposite direction connection, formed ariyoshi
Recombination.
The method is established on the basis of known protein sequence and function, can more efficiently be filtered out with better function
Functional protein.
The utility model has the advantages that
A kind of method of artificial reconstructed functional protein of the present invention, utilizes Secondary structure and important function
Energy structural region is that combination basic module constructs protein library, pass through sieve by the complete unordered random combine of intermodule
Choosing, orientation obtain the functional protein of more preferable efficiency.The literature data of the method building is huge, and function orientation is controllable, by right
The analysis of new functional protein structure understands effective module recombination and effective module in new functional protein, to understand
The structure function biological information and auxiliary of target protein improve design and rational protein and provide help.
Detailed description of the invention:
Fig. 1 P00552 secondary structure figure
Fig. 2 P00552 structural domain schematic diagram
Fig. 3 head and the tail sequence concentration optimizes, wherein 2000 DNA Marker of M.DL；1-3. different head and the tail sequence concentrations,
Head and the tail sequence concentration is followed successively by 50%, 10%, 2%
Fig. 4 re-assembly via PCR recurring number optimizes, wherein 2000 DNA Marker of M.DL；The reunion of 1-3. difference recurring number produces
Object, recurring number are followed successively by 3,5,10
Fig. 5 re-assembly via PCR template concentrations optimize, wherein 2000 DNA Marker of M.DL；1-4. different template concentrations weights
Poly- product, template concentrations are followed successively by 250ng/ μ L, 50ng/ μ L, 10ng/ μ L, 2ng/ μ L
Fig. 6 bacterium colony PCR electrophoretogram
Fig. 7 sequencing result simulation drawing
Specific embodiment:
A kind of method of artificial reconstructed kalamycin resistance albumen:
1. inquiry obtains the protein sequence in albumen database according to target enzyme protein structure, module, institute are chosen
Stating module is its secondary building unit (alpha-helix, beta-pleated sheet, β-corner), including important active site and ligand knot
Close site areas；
From kalamycin resistance albumen is inquired in EBI (European Bioinformatics research institute), the specific egg of structural information is searched
White matter.Its protein number is P00552, P00553, P05057.
The protein found is analyzed into its corresponding structure function in PDB (protein structures document data base)
Biological information.Corresponding module is found out, basic second grade structure (alpha-helix, beta-pleated sheet, β-corner including the protein
Deng) and important active site and ligand binding site.
P00552 amino acid sequence are as follows:
MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRPVLFVKTDLSGALNELQDEAARLS
WLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERA
RTRMEAGLVDQDDLDEEHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQD
IALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFFP00553 amino acid sequence are as follows:
MNESTRNWPEELLELLGQTELTVNKIGYSGDHVYHVKEYRGTPAFLKIAPSVWWRTLRPEIEALAWLD
GKLPVPKILYTAEHGGMDYLLMEALGGKDGSHETIQAKRKLFVKLYAEGLRSVHGLDIRECPLSNGLEKKLRDAKR
IVDESLVDPADIKEEYDCTPEELYGLLLESKPVTEDLVFAHGDYCAPNLI IDGEKLSGFIDLGRAGVADRYQDIS
LAIRSLRHDYGDDRYKALFLELYGLDGLDEDKVRYYIRLDEFFP05057 amino acid sequence are as follows:
MNGPIIMTREERMKIVHEIKERILDKYGDDVKAIGVYGSLGRQTDGPYSDIEMMCVMSTEEAEFSHEWT
TGEWKVEVNFDSEEILLDYASQVESDWPLTHGQFFSILPIYDSGGYLEKVYQTAKSVEAQTFHDAICALIVEELFEY
AGKWRNIRVQGPTTFLPSLTVQVAMAGAMLIGLHHRICYTTSASVLTEAVKQSDLPSGYDHLCQFVMSGQLSDSEKL
LESLENFWNGIQEWTERHGYIVDVSKRIPF
The secondary structure (see Fig. 1) of P00552 is analyzed, active site is located at No. 190 amino acid positions and isAspartic acid
(D), metal ion binding site is then in 195 and No. 208 amino acid positions, respectivelyAsparagine(N) andAspartic acid
(D), guarantee to contain enzymatic activity site and metal ion binding site as far as possible when module is chosen.
With P00552, objective function albumen chooses starting by analyzing its structural domain schematic diagram (see Fig. 2) as the main purpose
Sequence: 1-16, secondary structure: 23-27,35-40,47-52,59-72,79-86,89-97,90-97,98-101,109-123,
128-130、137-149、157-162、167-176、184-187、196-199、202-207、211-216、218-232、234-
244, active site and metal ion binding site: this 22 sections of different size of moulds of 189-196, termination sequence: 251-265
Block.
By analyzing P00553, P05057 secondary protein structure figure, active site region, i.e. albumen are chosen
The region 185-200 of the 143-157 of matter P05057, P00553.
24 different zones are had chosen altogether in summary as module.
Homing sequence: A (amino acid 1-16): MIEQDGLHAGSPAAWV
B (amino acid 23-27): DWAQQ
C (amino acid 35-40): AVFRLS
D (amino acid 47-52): LFVKTD
E (amino acid 59-72): ELQDEAARLSWLAT
F (amino acid 79-86): AVLDVVTE
G (amino acid 89-97): RDWLLLGEV
H (amino acid 90-97): DWLLLGEV
I (amino acid 98-101): PGQD
J (amino acid 1 09-123): PAEKVSIMADAMRRL
K (amino acid 1 28-130): PAT
L (amino acid 1 37-149): AKHRIERARTRME
M (amino acid 1 57-162): DLDEEH
N (amino acid 1 67-176): PAELFARLKA
O (amino acid 1 84-187): LVVT
Active site and metal ion binding site P (amino acid 1 89-196): GDACLPNI
Q (amino acid 1 96-199): IMVE
R (amino acid 202-207): RFSGFI
S (amino acid 211-216): RLGVAD
T (amino acid 218-232): YQDIA
U (amino acid 234-244): ATRDIAEELGG
Termination sequence V (amino acid 251-264): SQRIAFYRLLDEFF
P00553 active site sequence P3 (amino acid 1 85-200): HGDYCAPNLIIDGEKL
P05057 active site sequence P7 (amino acid 1 43-157): LFEYAGKWRNIRVQG
2. determining the genetic fragment of each module region
Selected tri- kinds of protein of P00552, P00553, P05057 are inputted into NCBI (US National Biotechnology Information
Center) in search corresponding gene order.Corresponding 24 gene orders of 24 modules are as follows, wherein A and V respectively headed by
Tailer sequence, P, P3, P7 active site and metal ion knot with tri- kinds of protein of P00552, P00553, P05057 respectively
Coincidence point region genetic fragment is corresponding, and the number P-A~P-PV, P-P3, P-P7 and the module sequence A~
V, P3, P7 are corresponded:
P-A、ATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTG(48nt)
P-B、GACTGGGCACAACAG(15nt)
P-C、GCCGTGTTCCGGCTGTCA(18nt)
P-D、CTTTTTGTCAAGACCGAC(18nt)
P-E、GAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACG(42nt)
P-F、GCTGTGCTCGACGTTGTCACTGAA(24nt)
P-G、AGGGACTGGCTGCTATTGGGCGAAGTG(27nt)
P-H、GACTGGCTGCTATTGGGCGAAGTG(24nt)
P-I、CCGGGGCAGGAT(12nt)
P-J、CCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTG(45nt)
P-K、CCGGCTACC(9nt)
P-L、GCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAA(39nt)
P-M、GAAGAGCATGATCTGGAC(18nt)
P-N、CCAGCCGAACTGTTCGCCAGGCTCAAGGCG(30nt)
P-O、CTCGTCGTGACC(12nt)
P-P、GGCGATGCCTGCTTGCCGAATATC(24nt)
P-Q、ATCATGGTGGAA(12nt)
P-R、CGCTTTTCTGGATTTATCGACATC(24nt)
P-S、CGGCTGGGTGTGGCGGAC(18nt)
P-T、TATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTT(45nt)
P-U、GGCGAATGGGCTGACCGCTTCCTCGTGCTTTAC(33nt)
P-V、TCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGA(45nt)
P-P3、CTGTTTGAATATGCAGGCAAATGGCGTAATATTCGTGTGCAAGGA(45nt)
P-P7、CACGGAGATTACTGTGCTCCGAACCTGATTATCGACGGTGAGAAGCTG(48nt)
3, by PCR or it is artificial synthesized in the way of at selected genetic fragment both ends Linker is added, form both ends
The single stranded DNA of Linker；
4, using the target enzyme protein sequence head and the tail genetic fragment as termination sequence, and only in described termination sequence one end
Linker is added, forms the single stranded DNA of one end Linker；
The Linker sequence are as follows: Linker1:5 '-AGGGAGTCCA-3’
Linker2:5 '-GGGAGTCCAT-3’
The Linker connects the corresponding amino acid sequence of gene order of two modules after digestion are as follows: G-S-P
The gene order that the Linker connects two modules after digestion is to encode the sequence of flexible amino acid,
Reduce influence of the Linker coding protein to zymoprotein space structure.
The synthesis of both ends Linker single stranded DNA and one end Linker single stranded DNA:
Linker1 sequence is added in other 23 ends of DNA sequence dna 5 ' in addition to first chain-ordering, what underscore indicated is limitation
Property endonuclease Hinf I recognition site, to guarantee that protection base is added in digestion simultaneously completely；By its in addition to tail chain sequence
Linker2 sequence, the identification position that it is restriction endonuclease Hinf I that underscore, which indicates, is added in 23 ends of DNA sequence dna 3 ' in he
Point, while addition protection base is designed, form the single stranded DNA of both ends Linker.It is connected after the Linker sequence digestion being added
Translation is formed " G-S-P " flexible amino acid sequence by the gene order of two modules.
General primer 5 '-ATGGACTCCC-3 ' is designed simultaneously, for forming double-stranded DNA except head and the tail sequence.Design head and the tail sequence
The complementary series of column: the complementary series of homing sequence is in 3 ' end design addition restriction enzyme sites and guarantor in addition to complementary pairing base sequence
Protect base, base sequence 5 '-CATATGCTCAGCTCG-3 ', thickened portion are last 3 base complementary with first chain, underscore
For the restriction enzyme site of Nde I；The complementary strand of tail end sequence or the complementary series of raw chains, and in 3 ' end designs of its raw chains
Restriction enzyme site and protection base, 5 '-TGA of base sequence is addedGAATTCCGG-3 ', last 3 alkali of chain headed by thickened portion
Base, underscore part are the restriction enzyme site of EcoR I.The upstream and downstream primer of specificity is designed simultaneously for expanding.
It is described that Nde I, I restriction enzyme site of EcoR and protection base formation protruding terminus is added in head and the tail sequence one end, guarantee
The connection of constructed gene library and carrier, while solving the problem of connection product sequence length in module connection regrouping process,
Guarantee that connection is not that unconfined connection is gone down.
5, under the action of polymerase, step 3 and step 4 gained single stranded DNA are synthesized into double-stranded DNA, use is restricted
Double-stranded DNA described in endonuclease digestion generates cohesive end, forms endonuclease bamhi；The restriction endonuclease is
I restriction endonuclease of Hinf.
Further, by the single-stranded utilization Klenow exo of the DNA of 22 both ends Linker-Polymerase is permanent at 37 DEG C
Temperature 4 hours, synthetic dsdna.Reaction system is as follows:
Head/tail single stranded DNA i.e. the single stranded DNA of one end Linker of the complementary pairing of synthesis, synthesis will be separately designed simultaneously
Double-stranded DNA.Reaction system is as follows:
One end Linker single stranded DNA (10-4M) 9.0μL
Head/tail complementary series (10-4M) 9.0μL
10×CutSmart Buffer 2.0μL
The constant temperature 4-5min at 95 DEG C guarantees that two complementary series form single straight chain, itself does not form any second level
Structure, slow cooling allow the single-stranded complementary pairing under certain salt ion environment of two DNA to form double-strand.
By corresponding 24 double-stranded DNAs of 24 modules under the effect of I restriction endonuclease of Hinf, same viscosity is cut out
End.
Further, for prevent there are also partially polymerized enzyme do not consume completely and by digestion generate cohesive end polishing at
For flat end, first described 24 double stranded DNA solutions, 75 DEG C of heated at constant temperature 20min, make Klenow exo-Polymerase complete deactivation,
It is slowly dropped to room temperature.
The polymerase and restriction endonuclease are all from the U.S. New England Biolabs (NEB) company, share same buffering
Solution can directly add I restriction endonuclease of Hinf of 0.4 μ L in DNA double chain solution, and constant temperature 6 is small at 37 DEG C
When, digestion generates cohesive end.
6, in the case where connecting enzyme effect, step 5 gained endonuclease bamhi is connected, forms library of recombinant；
Connect recombining reaction system:
16 DEG C of connections in metal bath are placed in stay overnight.Further, it is limited due to can still form Hinf I after connection recombination
Property endonuclease recognition site, to prevent remaining I restriction endonuclease of Hinf from cutting connection product again open
Come, inactivates I restriction endonuclease of Hinf 80 DEG C of heated at constant temperature 20min of the resulting endonuclease reaction solution of step 5, delay
Slowly it is down to room temperature.
The polymerase, restriction endonuclease and ligase are bought in NEB company, and buffer solution used removes the buffering of ligase
It is identical that solution contains other outer compositions of ATP, therefore need to only supplement part ATP and buffering when preparation connection recombining reaction system
Solution.
Further, in connection regrouping process, first by after first chain and 22 sequence Hybrid connections, tail chain is added, institute
Stating head and the tail sequence concentration ratio is 2%.
In the connection regrouping process, it should guarantee to obtain the disperse item comprising original kalamycin resistance gene length
Band guarantees that being formed by DNA includes two head and the tail sequences again.And the addition of head and the tail sequence can play termination connection in testing
Effect, therefore the concentration ratio of head and the tail sequence addition and sequencing all will affect the length of connection product.First by first chain and 22
After sequence Hybrid connections, tail chain is added.The concentration ratio of head and the tail module is optimized.Such as Fig. 3, by can be obvious in figure
Find out that generating cohesive end using restriction endonuclease connects, joint efficiency longer than A-T connection connection product genetic fragment
It is high.The head and the tail chain concentration ratio of final choice 2%.
Main sections product length after step 6 connection is less than the zymoprotein gene order length, passes through re-assembly via PCR
Extended；, the DNA fragmentation primer free after connection recombination is subjected to PCR reunion, and experiment condition is optimized.
As shown in Figure 4, under the premise of guaranteeing that reunion product is above target gene, 10 recurring numbers are enough.Meanwhile
DNA fragmentation template concentrations in re-assembly via PCR are optimized.
As shown in Figure 5, under the premise of guaranteeing that reunion product is above target gene, select DNA fragmentation in re-assembly via PCR dense
Degree is 250ng/ μ L, and reunion recurring number is 10 circulations.
7, the library of recombinant after connection is carried out PCR amplification, amplimer by the amplification and expression of the gene library
Restriction enzyme site is introduced, recycling is not less than the DNA fragmentation of original gene length areas, connect after digestion with carrier, express and screen.
It will be connected after genetic fragment double digestion after connection recombination with expression vector, the recombinant plasmid transformed sense after connection
By state cell and after expression and screening, 6 positive colonies are obtained, access 37 DEG C of shake culture 4h in 1mL Eppendorf pipe
Afterwards, bacterium solution PCR is carried out with vector primer, bacterium solution PCR product is subjected to electrophoresis and analyzed.
It will be appreciated from fig. 6 that the PCR product of different clones has different sizes, illustrate the DNA sequence dna in the mutant of building
Length is random distribution.
The amplification and expression of the gene library, by the library of recombinant after connection into PCR amplification, amplimer is introduced
Restriction enzyme site recycles the DNA fragmentation of original gene length areas, connect importing recipient bacterium with carrier after digestion and expresses and screen.
The validation verification of mutant library:
It is screened with kalamycin resistance gene mutant library of the method for resistance screening to building.It is dense using difference
The kanamycins of degree is screened.By repeatedly screening, 7 mutant with kalamycin resistance have been filtered out at present,
Highest resistance concentration is 1920ng/ μ L (64 times of as common kalamycin resistance).The effective mutant of picking is sequenced, and is surveyed
Sequence result is shown in Fig. 7.
Screening obtains the amino acid sequence of effective mutant:
1、A-R-B-O-P-M-T-C-O-P-M-D-K-K-F-I-S-K-K-B-N-D-J-S-C-V
MIEQDGLHAGSPAAWVGSPRFSGFIDIGSPDWAQQGSPLVVTGSPGDACLPNIGSPEEHDLDGSPYQDI
ALATRDIAEELGSPAVFRLSGSPLVVTGSPGDACLPNIGSPEEHDLDGSPLFVKTDGSPPATGSPPATGSPAVLDVV
TEGSPPGQDGSPRLGVADGSPPATGSPPATGSPDWAQQGSPPAELFARLKAGSPLFVKTDGSPPAEKVFMADAMRRL
GSPRLGVADGSPAVFRLSGSPSQRIAFYRLLDEFF
26 module recombinations, original P00552 active site and metal ion binding site containing there are two have 60ng/ μ L
Kalamycin resistance.
2、A-P-C-R-D-T-H-P-M-J-K-K-T-J-K-B-O-C-H-L-R-B-Q-N-V
MIEQDGLHAGSPAAWVGSPGDACLPNIGSPAVFRLSGSPRFSGFIDIGSPLFVKTDGSPYQDIALATRD
IAEELGSPDWLLLGEVGSPGDACLPNIGSPEEHDLDGSPPAEKVSIMADAMRRLGSPPATGSPPATGSPYQDIALAT
RDIAEELGSPPAEKVSIMADAMRRLGSPPATGSPDWAQQGSPLVVTGSPAVFRLSGSPDWLLLGEVGSPAKHRIERA
RTRMEGSPRFSGFIDIGSPDWAQQGSPIMVEGSPPAELFARLKAGSPSQRIAFYRLLDEFF
25 module recombinations, original P00552 active site and metal ion binding site containing there are two have 60ng/ μ L
Kalamycin resistance.
3、A-D-P7-F-H-Q-R-J-P-M-T-C-K-F-J-K-P-R-N-B-E-H-S-L-J-J-E-V
MIEQDGLHAGSPAAWVGSPLFVKTDGSPHGDYCAPNLIIDGEKLGSPAVLDVVTEGSPDWLLLGEVGSP
IMVEGSPRFSGFIDIGSPPAEKVSIMADAMRRLGSPGDACLPNIGSPEEHDLDGSPYQDIALATRDIAEELGSPAVF
RLSGSPPATGSPAVLDVVTEGSPPAEKVSIMADAMRRLGSPPATGSPGDACLPNIGSPRFSGFIDIGSPPAELFARL
KAGSPDWAQQGSPELQDEAARLSWLATGSPDWLLLGEVGSPRLGVADGSPAKHRIERARTRMEGSPPAEKVSIMADA
MRRLGSPPAEKVSIMADAMRRLGSPELQDEAARLSWLATGSPSQRIAFYRLLDEFF
28 module recombinations, original P00552 active site and metal ion binding site containing there are two, one original
P05057 active site, the kalamycin resistance with 60ng/ μ L.
4A-U-J-S-C-H-L-D-T-H-P-M-Q-S-J-T-P-M-Q-N-D-R-B-T-P-M-P3-B-T-V
MIEQDGLHAGSPAAWVGSPGEWADRFLVLYGSPPAEKVSIMADAMRRLGSPRLGVADGSPAVFRLSGSP
DWLLLGEVGSPAKHRIERARTRMEGSPLFVKTDGSPYQDIALATRDIAEELGSPDWLLLGEVGSPGDACLPNIGSPE
EHDLDGSPIMVEGSPRLGVADGSPPAEKVSIMADAMRRLGSPYQDIALATRDIAEELGSPGDACLPNIGSPEEHDLD
GSPIMVEGSPPAELFARLKAGSPLFVKTDGSPRFSGFIDIGSPDWAQQGSPYQDIALATRDIAEELGSPGDACLPNI
GSPEEHDLDGSPLFEYAGKWRNIRVQGGSPDWAQQGSPYQDIALATRDIAEELGSPSQRIAFYRLLDEFF
30 module recombinations, original P00552 active site and metal ion binding site containing there are three, one original
P00553 active site, the kalamycin resistance with 240ng/ μ L.
5、A-J-K-B-O-P7-U-N-D-B-M-S-K-O-D-I-T-Q-T-C-J-P-M-J-T-H-V
MIEQDGLHAGSPAAWVGSPPAEKVSIMADAMRRLGSPPATGSPDWAQQGSPLVVTGSPHGDYCAPNLII
DGEKLGSPGEWADRFLVLYGSPPAELFARLKAGSPLFVKTDGSPDWAQQGSPDWAQQGSPRLGVADGSPPATGSPLV
VTGSPLFVKTDGSPPGQDGSPYQDIALATRDIAEELGSPIMVEGSPYQDIALATRDIAEELGSPAVFRLSGSPPAEK
VSIMADAMRRLGSPGDACLPNIGSPEEHDLDGSPPAEKVSIMADAMRRLGSPYQDIALATRDIAEELGSPDWLLLGE
VGSPSQRIAFYRLLDEF
27 module recombinations, contain 1 original P00552 active site and metal ion binding site, one original
P05057 active site, the kalamycin resistance with 240ng/ μ L.
6、A-K-U-L-R-M-B-N-O-Q-C-P-D-G-E-H-F-G-R-S-T-U-I-J-V
MIEQDGLHAGSPAAWVGSPPATGSPGEWADRFLVLYGSPAKHRIERARTRMEGSPRFSGFIDIGSPEEH
DLDGSPDWAQQGSPPAELFARLKAGSPLVVTGSPIMVEGSPAVFRLSGSPGDACLPNIGSPLFVKTDGSPRDWLLLG
EVGSPELQDEAARLSWLATGSPDWLLLGEVGSPAVLDVVTEGSPRDWLLLGEVGSPRFSGFIDIGSPRLGVADGSPY
QDIALATRDIAEELGSPGEWADRFLVLYGSPPGQDGSPPAEKVSIMADAMRRLGSPSQRIAFYRLLDEFF
25 module recombinations, contain an original P00552 active site and metal ion binding site, have 240ng/ μ
The kalamycin resistance of L.
7、A-T-C-P-M-B-J-K-O-R-S-P3-U-B-E-U-R-F-N-L-P-M-R-D-H-V
MIEQDGLHAGSPAAWVGSPYQDIALATRDIAEELGSPAVFRLSGSPGDACLPNIGSPEEHDLDGSPDWA
QQGSPPAEKVSIMADAMRRLGSPPATGSPLVVTGSPRFSGFIDIGSPRLGVADGSPLFEYAGKWRNIRVQGGSPGEW
ADRFLVLYGSPDWAQQGSPELQDEAARLSWLATGSPGEWADRFLVLYGSPRFSGFIDIGSPAVLDVVTEGSPPAELF
ARLKAGSPAKHRIERARTRMEGSPGDACLPNIGSPEEHDLDGSPRFSGFIDIGSPLFVKTDGSPDWLLLGEVGSPSQ
RIAFYRLLDEFF
26 module recombinations, original P00552 active site and metal ion binding site containing there are two, one original
P00553 active site, the kalamycin resistance with 1920ng/ μ L.
Sequencing result is analyzed, module simulation such as Fig. 7 is carried out.The effective mutant obtained as can be seen from Figure is complete
Upset original gene sequence entirely, each effective mutant remains at least one original gene active site, and
Recombination module in mutant is at least 25 pieces, obtains new and with better function artificial protein, is opened broader
Sequence space provides help to be better understood by structure function and the biological information of protein.
Claims (5)
1. a kind of method of artificial reconstructed functional protein, it is characterised in that: based on target protein molecular structure, in protein
Its corresponding structure function biological information is analyzed in crystal structure document data base, constructs recombination module, and the module is it
Secondary building unit, and including important active site and ligand binding site region；It determines and encodes each module
Linker2 is added in 3 ' ends of first chain gene segment by genetic fragment, and Linker1, each centre is added in 5 ' ends of tail chain genetic fragment
Linker1 is added in 5 ' ends of genetic fragment, Linker2 is added in 3 ' ends, and carries out digestion, digestion using restriction endonuclease
Segment completely random connection combination building library of recombinant, expresses and screens；
The length that the Linker connects the gene order of two modules after digestion is 9 bases, and it is soft that G-S-P is formed after translation
Acidic amino acid sequence；The Linker gene order are as follows: Linker1:5 '-AGGGAGTCCA-3 ', Linker2:5 '-
GGGAGTCCAT-3’。
2. a kind of method of artificial reconstructed functional protein as described in claim 1, it is characterised in that: the block length is 3-
50 amino acid.
3. a kind of method of artificial reconstructed functional protein as claimed in claim 2, it is characterised in that: the block length is 3-
20 amino acid.
4. a kind of method of artificial reconstructed functional protein as described in claim 1, it is characterised in that the method includes following
Step:
(1) according to objective function protein structure, inquiry obtains the protein sequence in albumen database, in protein crystal
Its corresponding structure function biological information is analyzed in configuration data database, constructs recombination module, and the module is its second level
Structural unit, and including important active site and ligand binding site region；
(2) genetic fragment for encoding each module is determined；
(3) by PCR or it is artificial synthesized in the way of the 5 ' of selected other genetic fragments in addition to head and the tail genetic fragment
Linker1 is added in end, Linker2 is added in 3 ' ends, forms the single stranded DNA of both ends Linker；
(4) using the head and the tail genetic fragment in the objective function albumen coded sequence as termination sequence, in first chain gene segment
3 ' end be added Linker2, tail chain genetic fragment 5 ' end be added Linker1, formed one end Linker single stranded DNA；
(5) under the action of polymerase, single stranded DNA obtained by step (3) and step (4) is synthesized into double-stranded DNA, use is restricted
Double-stranded DNA described in endonuclease digestion generates cohesive end, forms endonuclease bamhi；
(6) in the case where connecting enzyme effect, endonuclease bamhi obtained by step (5) is connected, forms library of recombinant；
(7) amplification and expression of the gene library: the library of recombinant after connection is subjected to PCR amplification, amplimer draws
Entering restriction enzyme site, recycling is not less than the DNA segment of the functional protein original gene length areas, connect after digestion with carrier,
It expresses and screens.
5. a kind of method of artificial reconstructed functional protein as claimed in claim 4, it is characterised in that: after step (6) connection
Fragment products length be less than the functional protein gene order length, then by the fragment products primer free carry out PCR reunion.
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| CN101792934A (en) * | 2009-08-26 | 2010-08-04 | 青岛科技大学 | Novel method for building ultra-high capacity gene library based on combination principle and PCR |
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