CN1167151A - Method for deciding relative translation initial rate of external source genes in colibacillus - Google Patents

Abstract

The determination method of relative translation rate of exogenous gene in E. coli includes the following steps: making quantitative analysis and calculation for factors having influence on mRNA traslation initiation rate, including percentage of combination of energy and ribosome of secondary structure of translation initiation zone and formylmethionyl tRNA with ribosome combined in mRNA translation initiation zone, translation initiation delay rate and effective concentration of translation initiation zone, integrally considering relationship of the above-mentioned three calculated values and translation initiation rate and providing formula of relative translation initiation rate and method. According to this, the translation rate of protein can be determined, and according to this method, the mRNA translation initiation zone can be designed or improved so as to raise the expression quantity of exogenous gene in E. coli.

Description

The decision method of a kind of foreign gene relative translation initial rate in intestinal bacteria

The present invention relates to improve in the engineered intestinal bacteria system method of exogenous gene expression amount.

It has been a proven technique that intestinal bacteria are used for the producer gene engineering product as a kind of host bacterium.Adopt genetic engineering means exogenous genetic fragment can be cloned on the specific plasmid, import intestinal bacteria, obtain target protein, satisfy the different demands of people's production, life and healthy aspect by fermentation, separation and purification.But the speed that target protein is expressed in the intestinal bacteria system exists marked difference, and some gene fragment is easy to express, and other genes are considerably less at the product of expression in escherichia coli.At the expression in escherichia coli foreign gene, the height of its output depends primarily on the height of the speed of transcribing and translate two steps with genetically engineered.The height of translating a leg speed rate then depends primarily on the height of translation initial rate.In the intestinal bacteria initiating process of protein synthesis be small subunit ribosome and formylmethionyl tRNA and mRNA the interactional process in translation initiation district (TIR) (Gold, L.Ann.Rev.Biochem.1988,57,199-233).For many years, many laboratory studyes and analyzed the nucleotide sequence of a large amount of intestinal bacteria translation initiation sites and determined to influence among the TIR key character of translation initial rate.These features comprise: the distance between initiation codon, Shine/Dalgarno (SD) sequence, SD sequence and the initial code, the character of-3 bit bases etc., and carried out some quantitative analyses.Another important factor that influences translation initial rate is the secondary structure of mRNA, though many articles had been done many analyses and had been proposed various explanations to this, but still can't make quantitative analysis so far, we can say that this is one of biochemical and molecular biological difficult problem the influence of mRNA secondary structure.In recent years, the outstanding work that relates to the theory of translation initiation has two: first is the difference that de Smit and van Duin have analyzed the translation speed that the coat protein mRNA minority base that changes the MS2 phage causes, from chemical equilibrium derived the free generation of translation initiation complex can with relation (the de Smit of translation speed, M.H.and van Duin, J.Proc.Natl.Acad.Sci.USA 1990,87,7668-7672).Their conclusion is that the free generation of translation initiation complex can differ from 1.4 kilocalories/mol and will cause on the translation speed ten times difference.But because freely generating of translation initiation complex can can't be calculated, they do not solve the problem of quantitative Analysis.They can only replace with the difference between the energy of the secondary structure of the mRNA of a certain segment length translation initiation complex freely generate can difference, be like this can't quantitative comparison mRNA translation speed.Another work be Barrick etc. by random mutation studied different TIR structures to the influence of beta-galactosidase enzymes translation (Barrick, D.et al.Nucleic Acids Res.1994,22,1287-1295).They have calculated among the mRNA TIR relation that probability and translation speed appear in base on each position, and this is a kind of experimental formula.And he has only calculated-11 → 0 base, and in fact the TIR scope is much bigger.Therefore, going back the method that none can quantitatively judge translation initial rate so far, is the problem that remains further research.

The object of the invention provides the decision method of a kind of foreign gene relative translation initial rate in intestinal bacteria.This method is the quantitative analysis from rrna, formylmethionyl tRNA and mRNA translation initiation district's interaction energy, rrna is in conjunction with percentage, and a plurality of influence factors such as mRNA translation initiation retardation rate and mRNA translation initiation district effective concentration are taken all factors into consideration the method for calculation of the relative translation initial rate of mRNA of proposition.It can be used for the mRNA sequence is translated the judgement of speed, thereby judges the expression rate of foreign protein in the intestinal bacteria system.

A kind of foreign gene of the present invention relative decision method of translation initial rate in intestinal bacteria, it is by the method for calculation of the relative translation initial rate that rrna, formylmethionyl tRNA and the quantitative analysis of mRNA translation initiation district's interaction energy is proposed mRNA.The structure in mRNA translation initiation district, the secondary structure that comprises SD sequence, initiation codon and mRNA translation initiation district all influence the interaction of mRNA translation initiation district and rrna, formylmethionyl tRNA.3 of secondary structure energy needed, SD sequence and rrna 16S rRNA by taking the translation initiation district in 5 ' head to the mRNA of all lengths apart ' holds the quantitative analysis of the interactional energy of anticode of interactional energy, initial code and formylmethionyl tRNA to calculate influence factors such as the retardation rate of rrna and mRNA translation initiation district bonded percentage, mRNA translation initiation and mRNA translation initiation district effective concentration, and takes all factors into consideration the relative translation initial rate that these factors are calculated mRNA.Under all identical situation of promotor, encoding gene, host bacterium, culture condition, the difference of translation initial rate has determined the expression rate of this foreign gene in intestinal bacteria.Therefore, the present invention can be used for judging the expression rate of a kind of foreign gene intestinal bacteria.

Decision method of the present invention has been taken all factors into consideration rrna and mRNA translation initiation district bonded percentage, mRNA translation initiation retardation rate and these 3 factors that influence the mRNA translation initial rate of mRNA translation initiation district effective concentration.To calculate the quantitative analysis of these 3 factors below, the calculated value of these 3 factors and the relation of translation initial rate and thus the decision method of the relative translation initial rate of mRNA of proposition be elaborated.

One, translate while transcribing owing to mRNA, and have several rrna to translate usually on a mRNA, 5 ' head of mRNA is in continuous elongation.When exposed in mRNA translation initiation district, rrna just began combination with it, and began translation; Not combined mRNA then continues elongation along with the carrying out of transcribing or previous ribosomal translation, the secondary structure of the TIR of the mRNA of elongation can change, and taking TIR secondary structure energy needed in the mRNA of different lengths apart also may be different.Rrna is also just different with different lengths mRNA5 ' head bonded energy variation and probability.Therefore the speed of translation initiation is the overall result of the mRNA5 ' head effect of rrna, formylmethionyl tRNA (fMet-tRNA) and all lengths.

It is the first step of translation initiation that rrna (rRNA) interacts with mRNA translation initiation district (TIR), also is a most important step, can represent the translation initiation process of mRNA with it.This interaction is a chemical equilibrium:

The total concn that rRNA+TIR=TIR-rRNA establishes mRNA is [M], is F by rrna bonded mark, and unconjugated mark is (1-F), and rRNA concentration can be regarded a constant C as.The equilibrium constant $K=\frac{[\mathrm{TIR}-\mathrm{rRNA}]}{\left[\mathrm{rRNA}\right]\×\left[\mathrm{TIR}\right]}=\frac{\left[M\right]F}{C\left[M\right](1-F)}=\frac{F}{C(1-F)}$

F=CK ÷ (1+CK) is according to physical and chemical principle, G ° of the standard free energy of formation △ of TIR-rRNA binary complex=-RT 1nK

K=e ^{-△ G °/RT}, R=1.986 card/degree mol, T are absolute temperature.

In order to predict its translation speed according to the nucleotide sequence of mRNA, the present invention can decompose freely generating of translation initiation process according to the translation initiation process of mRNA in the intestinal bacteria, and replaces with the computable energy of computer.At first, the energy variation that takes place in TIR-rRNA binary complex generative process has the reduction △ G of SD sequence and 16SrRNA3 ' free energy when end combines _SDTake the TIR secondary structure energy needed △ G of mRNA apart _SNThere are fMet-tRNA anticode and initial code AUG bonded free energy to reduce △ G when after this forming the TIR-rRNA-fMet-tRNA ternary complex _I, and some other minor energy variation, as effect and the energy of thermal motion of molecule and the variation of potential energy etc. of ribosomal 530 rings with preceding 3 Nucleotide of initial code, these systems are designated as △ G _X△ G ° can be represented by the formula:

△ G °=△ G _SD+ △ G _SN+ △ G _I+ △ G _XCan calculate energy (Kierzek, R.et al.Biochemistry, 1986,25, the 7840-7846 that self is folded to form secondary structure in the energy variation of the base pairing between the nucleic acid and the nucleic acid molecule according to nucleic acid base paired thermodynamic data; Freier, S.M.et al.Proc.Natl.Acad.Sci.USA, 1986,83,9373-9377; Turner, D.H.et al.Annu.Rev.Biophys.Biochem.1988,17,167-192), for stable state energy, be designated as △ E by the energy that calculates like this.It and intravital truth are also inequality, are that parametric representation △ need multiply by coefficient, wherein △ E respectively for G ° with △ E _SDWith △ E _SNBe the main energy variation in this process, and △ E _SDTo different recons is variable, △ E _SNIn 5 ' head of the different lengths of each recon is variable.The present invention is placed on these two values in the variable.And initial code is identical for some foreign genes, △ E _IConstant.△ E _XValue then also do not have good method of calculation, the present invention is △ E _IWith △ E _XValue is placed in the not variable, that is:

△G°＝a(△E _SD+△E _SN)+b(△E _I+△E _X)

Like this, rrna can be expressed as in conjunction with the mark of mRNA TIR: $F=B{e}^{A({\mathrm{\ΔE}}_{\mathrm{SD}}+{\mathrm{\ΔE}}_{\mathrm{SN}})}\÷[1+B{e}^{A({\mathrm{\ΔE}}_{\mathrm{SD}}+{\mathrm{\ΔE}}_{\mathrm{SN}})}]$ A=-a/RT wherein, $B={\mathrm{Ce}}^{-b({\mathrm{\ΔE}}_I+{\mathrm{\ΔE}}_x)/\mathrm{RT}}$ 。The analysis of translation initial rate in intestinal bacteria draws A=0.508, B=18.5 to foreign gene according to the present invention.

Through above-mentioned processing, F has become △ E _SDWith △ E _SNFunction.△ E _SNThe difference with the variation of mRNA length (n), therefore, F also is the function of n, is designated as F (n).Its expression 5 ' head length is that the mRNA of n is by rrna bonded trend.When had just exposed in the translation initiation district of mRNA, the 5 ' head length n that establishes mRNA was w, and rrna bonded percentage is F _w, that not combined is 1-F _w, when mRNA length was elongated to arbitrary length n, rrna in conjunction with percentage was: P _n=F _n* (1-F _N-1(the 1-F of) * * _w).To the accumulative total rrna of the mRNA of all length in conjunction with percentage P _Always=∑ P _n

Change the different recons that the base in mRNA translation initiation district obtains, its secondary structure difference makes △ G _SNDifference also can make △ G because of having changed the SD sequence _SDDifferent.The result is that the rrna of each recon is in conjunction with the percentage difference.Rrna is high more in conjunction with percentage, and translation initial rate is just high more.

Two, the mRNA of different secondary structures is by rrna bonded percentage difference, the mean length difference of translation initiation consequently, promptly the translation initiation process of the mRNA that secondary structure is strong has been delayed, this delayed impact the speed of translation.The degree difference that the translation initiation of the mRNA of different secondary structures is delayed is represented with translation initiation retardation rate D (n).During n=w, D _w=1-P _w, when nRNA is stretched to n, be delayed the part be: D _n=1-∑ P _n, the translation initiation retardation rate D of accumulative total _Always=∑ D _nThe speed of this value and protein synthesis is inversely proportional to, and the recon translation initial rate that the translation initiation retardation rate is high more is just low more.

Three, mRNA also made by rrna bonded percentage difference-bar mRNA goes up in the rrna number difference of translating, i.e. the number of times difference that is utilized of each bar mRNA can be regarded as the difference of effective concentration of the TIR of mRNA.Ribosomal average headway on the mRNA is designated as u, and u equals the topped length 45 of rrna and adds that n-w and length are that the rrna of mRNA5 ' head of n combines percentage P _nThe sum of products.Be the u=∑ [(45+n-w) * P _n].MRNA5 ' holds the length=s of stop code, then the effective concentration of TIR [TIR]=[M] * s/u.The ratio of the effective concentration of each recon is: s/u.

Four, take all factors into consideration of the influence of above-mentioned factor, the present invention proposes the calculation formula of the relative translation initial rate of mRNA translation initial rate

P as can be seen from formula _AlwaysValue is big more to show that the TIR of mRNA and rrna combination rate are just high more, and the protein translation inception rate is also high more; D _AlwaysValue is big more to show that the translation initiation delay is many more, and the protein translation inception rate is low more; The big more rrna average headway that shows of u value is big more, and promptly mRNA effective concentration is low more, and the protein translation inception rate is low more.The variation of these three parameters is by the structures shape of the TIR of mRNA after all, that is to say mRNA TIR structure influence the speed of protein translation.Therefore, aforementioned calculation method of the present invention can be judged the translation speed of mRNA quantitatively, also promptly can judge expression of exogenous gene speed quantitatively.Because this calculating is the nucleotide sequence according to mRNA, therefore can be according to this method design or the translation initiation district that improves mRNA, to improve the expression amount of foreign gene in intestinal bacteria.

The present invention has set up a kind of method of judging foreign gene translation initial rate in intestinal bacteria.The invention has the advantages that: 1) the initial segmental interaction of mRNA of rrna and a kind of length is often only considered in Yi Qian analysis, and the initial segmental of this length m RNA determines it also is random.Yet, in vivo mRNA transcribe with translation be link coupled, translation is undertaken by a plurality of rrna on a mRNA again, so 5 ' head of mRNA is in elongation constantly, the length of the mRNA that acts on mutually with rrna and its secondary structure are changing.The energy itself of secondary structure that only calculates one section mRNA of some length is a defective in theory.The present invention has considered the overall result of rrna and various 5 ' length m RNA effect, and has proposed the speed that translation initiation retardation rate and these two physical quantitys of mRNA TIR effective concentration are used for calculating translation initiation.This is a new development for the translation initiation theory in the intestinal bacteria.2) the present invention reduces, takes apart the TIR secondary structure institute energy requirement of mRNA, the anticode of formylmethionyl tRNA and the free energy reduction of initial code effect etc. with the free energy that freely generating of translation initiation complex can be decomposed into SD and 16S rRNA effect, and calculate by nucleotide sequence with computer program, this makes us can judge the speed of translation initiation according to the nucleotide sequence of mRNA quantitatively.The embodiment of back shows that employing the present invention judges that method of calculation can better meet with experimental result for the calculation result of 5 ' PCNA-beta galactosidase enzyme fusion rotein.

The present invention is further elaborated by following examples, but does not place restrictions on scope of the present invention.

Embodiment 1,

Different sequencings of expressing the TIR of active people 5 ' PCNA-LacZ.Having obtained 269 by random mutation can be at different active people PCNA5 ' 34 amino acid-beta galactosidase enzyme α subunit fusion rotein (5 ' PCNA-LacZ) the recon of expressing of expression in escherichia coli.We have selected the recon of 7 different expression levels, and the difference up of its expression amount is more than 20 times.Measured the nucleotide sequence of this 7 recons 5 ' end with the terminal cessation method of the two deoxidations of Sanger.The result is as follows:

SD +1

A39：5′GGGA?AAGCUU?AUUAUA?GAGGU?AUAUUUU?AUG······3′

A42：5′GGGA?AAGCUU?UUAUAA?GAGGU?AUUCAUC?AUG······3′

B50：5′GGGA?AAGCUU?AUUAAA?GAGGU?AGCAAGA?AUG······3′

C28：5′GGGA?AAGCUU?CAAAC?GGAGGU?UAAGAUG?AUG······3′

B55：5′GGGA?AAGCUU?UGUGG?GGAGGU?ACUUCUU?AUG······3′

D13：5′GGGA?AAGCUU?CCACUA?GAGGU?UGCCACC?AUG······3′

114 nucleotide sequences of PCNA behind C59:5 ' GGGA AAGCUU GGACGC GAGGU UCAGGUU AUG3 ' AUG are identical, are connected correctly with the LacZ joint.The complete nucleotide sequence of 5 ' PCNA-LacZ fusion gene coding is as follows:

+1

5′AUG?UUC?GAG?GCG?CGC?CUG?GUC?CAG?GGC?UCC?AUC?CUC?AAG?AAG?GUG?UUG?GAG?GCA

CUC?AAG?GAC?CUC?AUC?AAC?GAG?GCC?UGC?UGG?GAU?AUU?AGC?UCC?AGC?GGU?GUA?AAC

CUG?CAG/UCG?AAU?UCA?CUG?GCC?GUC?GUU?UUA?CAA?CGU?CGU?GAC?UGG?GAA?AAC?CCU

←PCNA/LacZ→

GGC?GGU?ACC?CAA?CUU?AAU?CGC?CUU?GCA?GCA?CAU?CCC?CCU?UUC?GCC?AGC?UGG?CGU

AAU?AGC?GAA?GAG?GCC?CGC?ACC?GAU?CGC?CCU?UCC?CAA?CAG?UUG?CGC?AGC?CUG?AAU

GGC?GAA?UGG?CGC?CUG?AUG?CGG?UAU?UUU?CUC?CUU?ACG?CAU?CUG?UGC?GGU?AUU?UCA

+378

CAC?CGC?AUA?UGG?UGC?ACU?CUC?AGU?ACA?AUC?UGC?UCU?GAU?GCC?GCA?UAG

Stop code

Embodiment 2,

The calculating that the accumulative total rrna combination rate of each recon, accumulative total are translated retardation rate, rrna average headway.

According to the different IPs nucleotide sequence of 7 mRNA TIR among the embodiment 1, calculate G ° of △ according to nucleic acid base paired thermodynamic data, translate retardation rate and rrna average headway with △ G ° calculating accumulative total translation initial rate, accumulative total separately again.Their P _w→ ∑ P _W+4, and ∑ P _W+29, D _AlwaysAs follows with the numerical value of u: recon P _w∑ P _W+1∑ P _W+2∑ P _W+3∑ P _W+4∑ P _W+29D _Alwaysu

A39 0.8022 0.9609 0.9923 0.9985 0.9997 1 0.4443 47.6

A42 0.7940 0.9576 0.9913 0.9982 0.9996 1 0.4654 47.6

B50 0.7769 0.9502 0.9889 0.9975 0.9995 1 0.5102 47.6

C28 0.6071 0.8456 0.9393 0.9762 0.9906 1 1.0406 47.5

B55 0.5451 0.7931 0.9332 0.9620 0.9784 0.9998 1.2845 47.5

D13 0.2155 0.3845 0.5172 0.6212 0.7028 0.9017 6.5160 53.7

C59 0.2070 0.3712 0.5014 0.6046 0.6770 0.9168 6.7029 53.5

Embodiment 3

For the judgement of protein translation speed and with the comparison of measured value of experiment.

According to the P that is calculated among the embodiment 2 _Always, D _AlwaysFurther calculate protein translation speed with the numerical value of u, calculate and the translation speed of differentiating above-mentioned 7 recons is A39＞A42＞B50＞C28＞B55＞D13＞C59.And represent the activity of the beta galactosidase enzyme of fusion rotein with the ability of conventional mensuration hydrolysis p-nitrophenol phosphoric acid.Concrete with A ₄₂₀The ability of hydrolysis p-nitrophenol phosphoric acid in the colorimetric estimation cell pyrolysis liquid, with every milliliter of bacterium liquid, an A ₆₀₀Optical density(OD), per minute produces an A ₄₂₀It is a unit.By comparison, show that the activity of decision method provided by the present invention and practical measurement is consistence to two groups of data.The sliding property of the calculated amount of each recon and measuring is as follows:

Enzymic activity (unit) r * 10000 of the relative translation initial rate measuring that recon calculates

A39 0.047229 60.84 7.7628

A42 0.045105 49.51 9.1104

B50 0.041161 41.34 9.9566

C28 0.020244 23.46 8.6293

B55 0.016397 19.11 8.5805

D13 0.002576 2.89 8.9144

C59 0.002558 2.86 8.9447 is the enzymic activity of the relative translation initial rate ÷ measuring of r=calculating wherein.The relative deviation of calculated value and the ratio of experimental value is less than 7%.The back Figure 1 shows that the figure that the sliding property of the beta galactosidase enzyme of measuring is done the relative translation initial rate that calculates, and both meet to such an extent that be reasonable.

Claims (1)

1. the foreign gene decision method of translation initial rate relatively in intestinal bacteria, it is by the energy of intestinal bacteria mRNA translation initiation region two-stage structure and rrna, the interactional energy of formylmethionyl tRNA and mRNA translation initiation district are carried out quantitative analysis and judge the method for mRNA translation initiation plot structure for the influence of exogenous gene albumen translation initial rate, it is characterized in that this decision method is by to following three principal elements that influence translation initial rate:

(1) the secondary structure energy in mRNA translation initiation district and rrna, formylmethionyl tRNA combine percentage with the energy of mRNA translation initiation district effect and the rrna that is determined by these energy,

(2) mRNA translation initiation retardation rate,

(3) mRNA translation initiation district effective concentration is carried out quantitative analysis and is calculated, and with the calculation formula that above-mentioned three calculated values and relation between the translation initial rate propose the relative translation initial rate of mRNA is:

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