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Statistical optimization for IMAC purification of secreted hEPO

Statistical optimization for immobilized metal a?nity puri?cation

of secreted human erythropoietin from Drosophila S2cells

Hwa Sung Shin and Hyung Joon Cha *

Department of Chemical Engineering and Division of Molecular and Life Sciences,Pohang University of Science and Technology,

Pohang 790-784,Republic of Korea

Received 25September 2002,and in revised form 4November 2002

Abstract

We used a novel approach to a?nity purify human erythropoietin (hEPO)following its secretion from Drosophila melanogaster S2cells.Immobilized metal a?nity puri?cation of hEPO was optimized using a two-step serial statistical optimization strategy.After determining the elution conditions (based on preliminary batch-type puri?cation experiments),the ?rst optimization step considered three puri?cation factors;resin,equilibrium,and washing.The results of this analysis showed that the resin amount was the major factor in?uencing yield and purity in both model equations and the washing factor lowered the con?dence limits of the acquired model equations.The washing conditions were then set based on the results of the ?rst step optimization and the second step then optimized three factors;resin,equilibrium,and elution.The yield and purity of hEPO were then compared following puri?cation using three di?erent approaches;batch-type puri?cation based upon the conditions determined by serial statistical optimization,batch-type puri?cation performed in preliminary experiments,and FPLC column chromatography-type puri?cation.We found that the serial statistical optimization approach provided the best combination of yield and purity.These ?ndings indicate that serial statistical optimization strategies can be successfully employed for immobilized metal a?nity protein puri?cation using either batch-type or column approaches.

Keywords:Statistical optimization;Immobilized metal a?nity puri?cation;Human erythropoietin;Drosophila S2cells

Many types of recombinant expression systems have been used to produce heterologous proteins.Although puri?cation of such proteins is usually an important post-production step,it is generally di?cult to do,de-spite the development of a range of techniques.Puri?-cation di?culties led to the development of immobilized metal a?nity puri?cation,which was ?rst used by Porath and co-workers in 1975[1].This method has been successfully employed for puri?cation of polyhis-tidine-tagged proteins,mainly due to its simplicity,high selectivity,and large capacity [2].The hexa histidine tag eHis T6assists foreign proteins in binding to an im-mobilized metal a?nity resin that is chelated with nickel-nitrilotriacetic acid (Ni-NTA).Being small and uncharged,the tag does not generally interfere with secretion or folding of expressed foreign proteins.

Production of His-tagged proteins has been successfully performed in several expression systems,such as Escherichia coli [3],yeast [4],insect [5],and mammalian cells [6].

The ultimate objective of protein puri?cation is to achieve both high yield and purity.Like other tech-niques,the e?ciency of immobilized metal a?nity pu-ri?cation can be a?ected by factors including the natural properties of the target protein,culture conditions,and puri?cation conditions [7].To obtain both high yield and purity,it is important to understand the relation-ship between these two goals and the puri?cation factors,and to optimize puri?cation conditions ac-cordingly.Often optimization of a?nity puri?cation procedures has been performed crudely,involving merely adjusting the quantity of metal-chelated resin,the concentration of imidazole in the washing and elu-tion bu?er,and so on [8].However,this approach may not account for interactions among puri?cation

factors,

Protein Expression and Puri?cation 28(2003)

331–339

https://www.wendangku.net/doc/655995507.html,/locate/yprep

*Corresponding author.Fax:+82-54-279-2699.E-mail address:hjcha@postech.ac.kr (H.J.Cha).

which can a?ect optimization.A statistical approach, such as response surface methodology(RSM),can be used as an alternative optimization tool.RSM based on factorial experiments is a statistical method of examin-ing the e?ect of test variables on measured responses [9,10].The mathematical model for RSM was derived from orthogonal polynomial?tting techniques.The RSM approach to optimization requires?rst an exper-imental design and then?tting experimental data into an empirical model equation to determine the optimum conditions.

eHisT6-tagged proteins have been successfully ex-pressed in insect cells,usually using a baculovirus system [11–13].However,this is a lytic system and may not be suitable for proteins that require post-translational modi?cations,as the secretory pathway apparatus may be damaged[14].Using Drosophila melanogaster S2 cells,a plasmid-based,and therefore non-lytic,expres-sion system has been developed for secretion of func-

tional human proteins[15].A number ofeHisT

6-tagged

foreign proteins have been successfully expressed and puri?ed using this system[16,17].In the present study, we expressed human erythropoietin(hEPO)in Dro-sophila S2cells.hEPO,with165amino acids and a molecular weight of approximately30kDa[18],is a glycoprotein,which is a principal growth factor re-sponsible for stimulation of proliferation and di?eren-tiation of responsive bone marrow erythroid precursor cells into more mature erythrocytes[19].

In the present work,we performed small-scale batch-type a?nity puri?cation in microcentrifuge tubes(the so-called protein mini-preparation)in order to easily manipulate a variety of puri?cation conditions.The objective of this work was to optimize immobilized metal a?nity protein puri?cation using statistical methodology.This was achieved by examining the ef-fects of several factors on a?nity puri?cation.To our knowledge,the present work demonstrates for the?rst time the successful application of statistical optimization to immobilized metal a?nity puri?cation methodology. Materials and methods

Cell culture and transfection

Drosophila S2cells(Invitrogen)were grown at27°C in M3medium(Shields and Sang M3insect medium; Sigma,St.Louis,MO)containing10%IMS(insect medium supplement;Sigma).Cell number was counted from each sample using a hemacytometer(Fisher Sci-enti?c,Pittsburgh,PA)and viability was determined by trypan blue(Sigma)exclusion using a0.4%(w/v)solu-tion.

S2cells were transfected with pMT/BiP/hEPO[20] that contains the Drosophila metallothionein promotor (pMT)that is activated by Cu2t,BiP signal sequence to

facilitate secretion,and hEPO cDNA,using lipofectin (Life Technologies,Grand Island,NY)for stable ex-pression.S2cells were plated in6-well culture plates at 2?106cells/mL,and after24h,cells were washed twice with M3medium and then left in2mL M3medium for 2h.Solutions containing the expression vector pMT/ BiP/hEPO(1l g)and selection vector pCoHygro(invi-trogen)(1l g),or lipofectin(10l g)were prepared sepa-rately in0.1mL M3medium,incubated for15min,and then mixed together for45min at room temperature. The vector mixture was added to0.8mL M3medium for a total volume of1mL and the solution was added to the cells.After24h,transfection medium was replaced with3mL M3medium supplemented with10%IMS and the cells were transferred into35-mm cell culture dishes.Hygromycin B(Sigma)(300l g/mL)was added to the medium to select transfected cells and every5 days cells were replenished with2mL M3medium/10% IMS/hygromycin.About2weeks later,colonies resis-tant to hygromycin B were observed and heterogeneous cell lines expressing hEPO were obtained after3–4 weeks.All colonies from one plate were pooled as a transfected cell line due to the di?culty in successfully selecting a single colony from a plate for expansion.

To produce secreted hEPO from the developed S2cell lines,cells were grown to1?106cells/mL(over95% viable)in M3medium/10%IMS/hygromycin using three 100-mm cell culture dishes and transferred into a 500mL spinner?ask(Wheaton,Millville,NJ)contain-ing250mL serum-free M3medium.Cells were incu-bated at27°C with constant stirring(80rpm)until a cell density of at least4?106cells/mL was reached,after which copper sulfate was added(500l M)to induce. Preliminary immobilized metal a?nity puri?cation of secreted hEPO

Two days after the induction ofeHisT

-tagged hEPO expression,culture medium was clari?ed by centrifuga-tion at5000rpm for5min and concentrated by ultra?l-tration using a10,000molecular weight cut-o?membrane(MWCO)(Amicon stirred cell,Model8400; Millipore,Bedford,MA)at4°C.Ni-NTA agarose resin (Cat.No.30210,50%suspension in30%ethanol,pre-charged with Ni2t;Qiagen,Valencia,CA)was used for

immobilized metal a?nity puri?cation ofeHisT

-tagged hEPO.Copper sulfate,the inducer of protein expression, was removed from the concentrated media by dialyzing twice against200volumes of bu?er A(50mM NaH2PO4, pH8,and300mM NaCl)overnight using12,000–14,000 MWCO tubing(Spectrum Laboratories,Rancho Do-minguez,CA),to prevent Cu2tfrom interfering with the

binding ofeHisT

-tagged hEPO to Ni-NTA agarose. After dialysis,200l L of this?sample solution?(contain-ing7:47l g=l L hEPO)and40l L Ni-NTA agarose beads

332H.S.Shin,H.J.Cha/Protein Expression and Puri?cation28(2003)331–339

(therefore,5.35l L resin per1l g hEPO)were equili-brated in a1.5-mL microfuge tube with1mL bu?er A containing1mM imidazole to reduce non-speci?c bind-ing of proteins and then mixed for5h.To elute bound proteins,a?nity beads were washed with bu?er A(1mL) containing0,1,5,10,20,50,80,or200mM imidazole.

Statistical optimization by response surface methodology

Optimization for a?nity puri?cation of the concen-trated and dialyzed hEPO was performed by RSM.In general,the form of the true response function cannot be known.Therefore,it is important to develop an ap-propriate approximation for the function.Special types of experimental design and suitable model equations were very important in this work.Examinations of all experimental conditions are very di?cult and ine?ective. Therefore,we employed a factorial experimental design called a central composite design(CCD)[9,21].There are many experimental designs for RSM—CCD,Box-Behnken design(BBD),Small composite design(SCD), and Plackett-Burman design(PBD).However,CCD is an e?cient design that is ideal for sequential experi-mentation and allows a reasonable amount of infor-mation to test lack of?t while not involving an unusually large number of design points.Actually,CCD is the most popular class of second-order design.We speci?ed the ranges on the design variables and per-formed experiments within these regions because it was thought that optimal condition might exist within the restricted regions and the design was known to be useful for any number of design variables.The center point of the design was repeated four times in order to allow a better estimate of the experimental error and to provide extra information.The experimental conditions and re-sults are summarized in Table1.One of the most useful models for approximating a region of a multifactor re-sponse surface is the polynomial model.Therefore,we used a second-order model that can be written like Eq.

(1)because it can take on a wide variety of functional forms that can work well as an approximation to the true response surface

Y?b0t

i?1

b i x it

i?1

b it3x2

tb7x1x2tb8x1x3

tb9x2x3tb10x1x2x3;e1Twhere Y is the response associated with experiments,x i is the parameter,and b i is the coe?cient.The values of coe?cient b i were calculated using SAS software(SAS Institute,North Carolina,USA)[22].Generally,a multiple linear regression analysis considers many independent variables that can a?ect the value of the dependent variable.Although considering many inde-pendent variables in a regression model that can e?-ciently describe changes to the dependent variable,it can also produce a strong linear relationship between independent variables.This phenomenon is called a multicollinearity,which creates serious problems in presumption and examination of regression analysis. First,when adding or eliminating an independent vari-able,the regression parameter estimates of other inde-pendent variables vary greatly.Second,the variances of the parameter estimates diverge and con?dence falls.

Table1

Experimental set for each level of three considering factors for puri?cation of secreted hEPO a

Num R E W b/L c Yield Purity

Step I Step II Step I Step II

1))) 1.20.8570.230.469

2))+0.880.8800.350.5

3)+)0.850.7080.290.5

4)++0.790.7180.270.474

5+))0.320.5610.0780.260

6+)+0.340.5000.190.284

7++)0.320.4640.150.313

8+++0.590.6480.240.369

9)1.68000.990.8250.230.264

0+1.68000.480.5930.200.323

110)1.6800.590.7380.330.434

120+1.6800.890.7420.480.615

1300)1.68 1.100.7860.180.353

1400+1.68 1.600.8000.480.706

15000 1.100.7860.420.627

160000.670.7440.530.697

170000.570.6000.410.612

180000.840.6460.290.387

a R;à?28:2;0?55,+?81.8;E;à?2:02;0?5,+?7.98;W;à?6:07;0?15,+?23.93;and L;à?90:2;0?150,+?209.8.

b For step I optimization.

c For step II optimization.

H.S.Shin,H.J.Cha/Protein Expression and Puri?cation28(2003)331–339333

Third,although the F-test results regarding the rela-tionship between independent variables and the depen-dent variable are statistically sound,those for individual parameters of important independent variables happen to have no physical meaning.Therefore,it is important to create an optimal regression model that minimizes the loss of information,and is simpli?ed by selecting ap-propriate independent variables a?ecting the dependent variable.In this study,the model equations were de-termined by two methods provided in the SAS software (explained in Results and discussion).Also,the maxi-mum responses of these models were solved using Lindo Callable software(Lindo Systems,Chicago,USA). Immobilized metal a?nity chromatography with gradient elution

Immobilized metal a?nity chromatography(IMAC)1 was performed using an FPLC system(Acta Prime Pu-ri?cation System;Amersham Biosciences,Uppsala, Sweden)at room temperature with1mL/min?ow rate.

A Hi Trap Chelating HP(Amersham Biosciences)af-?nity column(5mL volume)was charged with5mL of 0.1M NiSO4.The column was equilibrated with10mL bu?er A supplemented with10mM imidazole and1mL sample solution(derived from cell medium)was then loaded.The sample-loaded column was washed and eluted with bu?er A,incorporating an imidazole gradi-ent up to500mM.

SDS–PAGE analysis

The eluted fraction was mixed with SDS sample bu?er(10%sodium dodecyl sulfate(SDS),10%b-mercaptanol,0.3M Tris–HCl(pH6.8),0.05%brom-ophenol blue,and50%glycerol),boiled for5min,and resolved by12.5%SDS–polyacrylamide gel electropho-resis(SDS–PAGE).Then,hEPO bands were detected by silver staining(Bio-Rad,Hercules,CA).The stained gel was scanned and the digitized image was stored and analyzed by Gel-Pro Analyzer software(Media Cyber-netics,Silver Spring,MD).

Results and discussion

Preliminary a?nity puri?cation of secreted hEPO To employ a statistical approach for optimization of hEPO a?nity puri?cation,it was necessary to?rst gain information about puri?cation factors and the nature of the experiments.Therefore,preliminary a?nity puri?-cation was performed in which the sample solution (containing hEPO)was added to40l L Qiagen Ni-NTA a?nity resin(equivalent to5:35l L=l g-hEPO)and bound hEPO was eluted with imidazole.Fractions were analyzed using silver-stained SDS–PAGE(Fig.1).We found that the majority of total protein remained un-bound and was washed from the resin in0mM imid-azole bu?er(lane1).More proteins and unbound(or weakly bound)hEPO were eluted in5mM imidazole bu?er(lane2),with these proteins also representing those exceeding the binding capacity of the a?nity resin. hEPO(56.4%pure;lane5)and other proteins were eluted in50mM imidazole.A more puri?ed hEPO fraction(78.2%,lane6)was obtained following elution with80mM imidazole,while the remaining hEPO was eluted with200mM imidazole(lane7).

Experimental designs for statistical optimization The more experimental factors selected for RSM,the more accurate the empirical model equation.However, too many factors can create a more complicated model equation,resulting in an increase in the number of ex-periments to be performed.In some cases,experimental error increases as both the number of experiments and the time required increase.Therefore,it is important to select an appropriate number of critical puri?cation factors in an experimental design.Characteristics of a protein such as size,pI,and structure can largely a?ect its puri?cation.However,there are some experimental limits to analyze their impacts by using only one type of protein and RSM.To achieve this analysis,we need to check many proteins having diverse characteristics.For example,18di?erent proteins might be needed to in-vestigate impacts of the above three characteristics. Therefore,due to di?culty in showing the optimization guideline that re?ects characteristics of protein itself,we tried to optimize a?nity puri?cation of only one target protein,eHisT

-tagged hEPO.Also,there might be many considerable puri?cation factors such as bu?er compo-

1Abbreviations used:R,quantity(l L)of resin;E,imidazole concentration(mM)for equilibrium bu?er;W,imidazole concentra-tion(mM)for washing bu?er;L,imidazole concentration(mM)for elution bu?er;Y,relative yield;P,purity;DF,degree of freedom; IMAC,immobilized metal a?nity

puri?cation.Fig.1.SDS–PAGE analysis for preliminary immobilized metal a?nity puri?cation of secreted hEPO from https://www.wendangku.net/doc/655995507.html,ne M,molecular weight standard;lane1,0mM imidazole;lane2,5mM imidazole;lane3, 10mM imidazole;lane4,20mM imidazole;lane5,50mM imidazole; lane6,80mM imidazole;and lane7,200mM imidazole.

334H.S.Shin,H.J.Cha/Protein Expression and Puri?cation28(2003)331–339

sition,puri?cation temperature,a?nity resin,and im-idazole.In the case of bu?er composition,optimization of salt composition is a hard work because types and amounts of salt are very diverse.Also,the optimal bu?er conditions might di?er according to each sample pro-tein.Therefore,for an experimental convenience,we followed the bu?er composition that is recommended by the company supplying resin.In the case of puri?cation temperature,proteins are generally handled at low temperature to maintain their biological activity and structure.Therefore,we performed overall experiments in an ice-based chamber.In the present study,we se-lected resin quantity and imidazole concentration in bu?ers(equilibrium,washing,and elution)as consider-able factors that can greatly in?uence each a?nity pu-ri?cation step.

The preliminary a?nity puri?cation(Fig.1)provided important information regarding each puri?cation fac-tor and this information was used in statistical optimi-zation experiments.Because much hEPO remained unbound when using40l L a?nity resin,the range for resin quantity was set at10–100l L.Most non-speci?-cally bound proteins were eluted between5and20mM imidazole.Although some non-speci?cally bound pro-teins also eluted at50mM,these did not appear to be new proteins,but the same as those eluted between5 and20mM imidazole.Given a large amount of hEPO eluted with50mM imidazole,the imidazole concentra-tion range for the washing bu?er was set at0–30mM. The concentration of imidazole in the equilibrium bu?er regulates the amount of non-speci?c binding and1mM imidazole was used in the preliminary experiment.A great deal of non-speci?cally bound protein eluted at 5mM imidazole,along with large amounts of unbound hEPO(lane2,Fig.1).The80mM imidazole fraction contained some non-speci?cally bound proteins and the amount of hEPO eluted with10and20mM imidazole was insigni?cant.These observations indicate that the concentration of imidazole in the equilibrium bu?er could be higher without much loss of hEPO.Accord-ingly,a0–10mM concentration range of imidazole in the equilibrium bu?er was chosen.

We employed a two-step optimization strategy,with each step considering three puri?cation factors,as considering four factors would greatly increase the number of experiments required.The three puri?cation factors considered in the?rst optimization step were resin amount and imidazole concentrations in the equilibrium and washing bu?ers.The imidazole con-centration in the elution bu?er was?xed at80mM in order to guarantee hEPO elution.In addition,since no new non-speci?c bands appeared at the highest imid-azole concentration(200mM)in the preliminary ex-periment,it was expected that relative values of yield and purity for hEPO might not vary between80and 200mM imidazole.

After gaining an understanding of the relationship between related independent factors and the dependent factor for yield or purity in the?rst optimization step (data presented below),the second optimization step was performed.We?xed one factor that did not have a signi?cant in?uence on immobilized metal a?nity puri-?cation and performed RSM optimization for the re-maining three puri?cation factors,this time including elution.In the preliminary experiments,puri?ed hEPO appeared at50mM imidazole and more was eluted at 80mM.However,not much hEPO eluted at200mM, and since little hEPO appeared to elute at less than 50mM imidazole,the imidazole concentration range in the elution bu?er was set at50–250mM(the second optimization step data are presented below).

For the two-step statistical experiments,a central composite design that is widely used for?tting second-order model equations was employed.That comprises2k factorial points,2k axial points,and four center runs. The axial value a,depending on the operability of the experiment,was chosen as 1.68,like in many other statistical experiments.The experimental designs for the two-step optimization are shown in Table1. Statistical optimization of hEPO a?nity puri?cation:?rst step

For the?frst optimization step we performed18ex-periments following the experimental design(Fig.2A). Yield of puri?ed hEPO was calculated as a relative value based on the25kDa protein band of the standard mo-lecular weight marker(lane M,Fig.2).The values of relative puri?cation yield and purity of secreted hEPO are shown in Table 1.We used and compared two methods provided in the SAS software to?nd the best regression model equation to?t the experimental results. In the?rst method,we identi?ed all possible regression models according to the number of independent vari-ables and then selected the models that had maximal or adjusted R2,and the best regression model was

then Fig.2.SDS–PAGE analysis of puri?ed hEPO by immobilized metal a?nity puri?cations under18di?erent puri?cation conditions for(A)?rst and(B)second optimization https://www.wendangku.net/doc/655995507.html,ne M,molecular weight standard;lane1–18,di?erent conditions(shown in Table1).

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chosen.The second method involved stepwise regression whereby variables were selected or eliminated one by one by evaluating the relative importance of already or newly included variables into a model equation [23].In the ?rst step of the optimization experiment,we tried to ?nd meaningful equations regarding yield and purity within each e?ective limit (Tables 2and 3).We obtained two equations for yield and purity with F-test over 2.74and P values below 0.05for parameter estimates of in-dividual variables,as well as whole equations.The two equations showed that resin amount (R )was a major factor in?uencing yield and purity in both model equa-tions.Also,imidazole concentration of the equilibrium bu?er (E)was a meaningful variable in the purity equation,although E did not appear in the yield equa-tion (Table 3).However,imidazole concentration in the washing bu?er (W )was,if anything,only slightly related to the yield equation (Pr >j t j ,0.0695)and less related to the purity equation (Pr >j t j ,0.1222),causing less con-?dence (higher error limit)in model equations.Based on these data,we predicted that W might act negatively on

the second step experiment,thus the washing term was eliminated for that experiment.

Statistical optimization of hEPO a?nity puri?cation:second step

In the preliminary experiment,few non-speci?c pro-teins eluted at 20mM,but many eluted at 50mM.Therefore,for the second step optimization experiment,the washing bu?er contained 30mM imidazole,which was the upper limit of the concentration range in the ?rst step experiment,since neither yield nor purity is likely to be negatively a?ected at that concentration.Finally,we selected the resin amount and the imidazole concentrations in the equilibrium and elution bu?ers as related variables and the same approach as the ?rst step was applied.As shown in Table 2,the experimental equations re?ected their related experimental data e?-ciently within 10%con?dence limits.The coe?cients and their con?dence limits of all independent variables for the equations of yield and purity are displayed in

Table 2

Analysis of variance Source DF Sum of squares Mean square F value Pr >F

Model

Step I Yield 3 1.119370.37312 6.190.0067

Purity 40.176960.044248.440.0014Step II

Yield 40.173490.04337 5.830.0065Purity 30.210250.070085.970.0077

Error

Step I Yield 140.843870.06028——Purity 130.068120.00524——Step II

Yield 130.096740.00744——Purity 140.164380.01174——Corrected total

Step I Yield 17 1.96324———Purity 170.24508———Step II

Yield 170.27023———Purity

0.37462—

—

Table 3

Parameter estimate Experiment Variable Parameter estimate

Standard error

t Value

Pr >j t j

Step I

Intercept 1.278470.19999 6.39<0:0001Yield

W )0.051420.02616)1.970.0695R 2)0.000075790.00002193)3.460.0039W 2

0.001920.00083611 2.300.0377Intercept 0.962410.0693813.87<0:0001E )0.030400.01235)2.460.0286Purity

R 2)0.000036870.00000801)4.600.0005RW )0.000121050.00007322)1.650.1222REW 0.000029280.00001266 2.310.0377Step II

Intercept )0.305890.18489)1.650.1220R 0.011450.00374 3.060.0091Yield

L 0.004680.002052.290.0395R 2)0.000117340.00003310)3.550.0036L 2

)0.000012310.00000669)1.840.0889I

ntercept 0.207000.120851.710.1088R 0.012420.00469 2.650.0190Purity

R 2)0.000140620.00004096)3.430.0040

REL

0.000001668:990112E à7

1.850.0853336

H.S.Shin,H.J.Cha /Protein Expression and Puri?cation 28(2003)331–339

Table 3.All independent variables were also e?ective within about 10%con?dence limits.Optimal conditions for yield or purity of immobilized metal a?nity puri?-cation were determined from each second-step model equation using Lindo Callable software (Table 4).

Using SDS–PAGE analysis,we compared puri?ca-tion based on the second step optimization with those from both the preliminary experiment and FPLC-based immobilized metal a?nity chromatography (IMAC)using an elution gradient up to 500mM imidazole (Fig.3).The comparison showed that conditions derived from the serial statistical optimization strategy provided a competitively higher hEPO yield (Table 5).In terms of purity,IMAC exhibited the best result (93%),followed by the statistical approach and then the preliminary experiment.The statistically optimized puri?cation strategy resulted in the best combination of yield (42%)and purity (90%)of hEPO when the purity equation was employed for a?nity puri?cation.As shown in Table 4,in the experimental conditions from the second step

optimization,values of optimal R and L from the purity equation were higher than those from the yield equa-tion,while other factors (E and W )were the same.If this di?erence led to the above puri?cation results,we can surmise that higher yield and purity can be obtained from higher R and L values.However,although the optimal conditions from the yield equation produced a yield lower than that from the purity equation,we cannot conclude that the determined yield equation is incorrect.Also,we cannot conclude that the yield equation does not re?ect the experimental data well because the yield di?erence was not signi?cantly large.Most non-speci?cally bound proteins eluted at the low imidazole concentration (30mM),as shown in Fig.1.Therefore,although varying the imidazole concentra-tion from 190mM in the yield equation to 250mM in the purity equation,purity might be enhanced merely due to increased yield.The data presented in Fig.4show that neither yield nor purity was greatly a?ected if the elution bu?er di?ered between 190and 250mM imid-azole while they were highly dependent on the resin amount used in these studies.

Each sample to be puri?ed has its own salt concen-tration,quantity of non-speci?c binding protein,and/or degree of non-speci?city.Sample properties are highly a?ected by host cells and their growth media.If hEPO is expressed (or secreted)in other host cells,such as bacteria or mammalian cells,puri?cation e?ciency might be sig-ni?cantly di?erent from that using Drosophila S2cells.Also,characteristics of a?nity resin,such as binding ef-?ciency and puri?cation capacity,depend highly upon the manufacturers.Therefore,the optimal equation re-?ecting the puri?cation data must be varied depending on foreign protein sample,a?nity resin type,and so on.However,the relationship between the many

factors

Fig.3.SDS–PAGE analysis of puri?ed hEPO by several a?nity pu-ri?https://www.wendangku.net/doc/655995507.html,ne M,molecular weight standard;lane 1,preliminary immobilized metal a?nity puri?cation;lane 2,immobilized metal af-?nity puri?cation using optimal yield conditions;lane 3,immobilized metal a?nity puri?cation using optimal purity conditions;and lane 4,immobilized metal a?nity chromatography with gradient elution.

Table 5

Summary of comparison for several a?nity puri?cations Puri?cation step Volume (mL)

hEPO (l g)

Total protein (l g)

Yield (%)Purity (fold)Purity (%)Culture medium

250591.28883.2100 1.015

Ultra?ltration/Dialysis 15560.58407.594.8 1.015Preliminary puri?cation

15130.5174.022.1

4.2

Puri?cation using optimal condition from step II

Yield 15222.8247.537.75.586Purity 15247.5298.541.9 6.090IMAC puri?cation with gradient elution

15126.0135.821.3 6.293

Table 4

Experimental conditions for immobilized metal a?nity puri?cation Experiment R (l L)

E (mM)W (mM)L (mM)Preliminary 40(5:35l L =l g-HePO)12080Step II

Yield 48.79(6:53l L =l g-hEPO)1030190.1Purity

58.92(7:89l L =l g-hEPO)

250

H.S.Shin,H.J.Cha /Protein Expression and Puri?cation 28(2003)331–339

337

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The authors acknowledge the support for ful?lment of this work by POSTECH BSRI research fund,Yuhan Corp.research grant,and the Brain Korea 21program issued from the Ministry of Education,Korea.References

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win8.1系统安装详细图文教程

系统安装方式目前有三种，分别是硬盘装系统、U盘装系统、光盘装系统。它们各有优缺点小编就不在此累述。小编编写此篇教程的目的是为了教大家在系统没崩溃的情况下，通过硬盘安装GHOST系统的方式，实现快速装机目的。具体步骤如下：硬盘装系统一、系统下载完成之后，右键单击ISO镜像，弹出菜单选择解压文件；二、解压完成，文件夹内容如下，双击打开autorun.exe文件或直接打开名为”安装系统”的程序：

三、弹出的“AUTORUN.EXE”运行界面中选择“安装GHOST系统到C盘”；四、进入系统安装界面，如下图点选相应选项，点确认即可

选择完成，点击确定，然后选择自动重启，即可进入自动装机状态，时间约持续5~10分钟；注意事项：1、如果自动装机完成后没有直接进入桌面，而出现了黑屏状态，毋须担心，不是系统问题，直接手动重启电脑，重启完成即可正常使用。 2、解压时请直接进行解压，不可系统存放路径不可存有中文字符，否则将无法正常安装。 3、请将解压出的gho文件放到除C盘外的分区，否则将无法正常安装；点击确定后会重新启动自动化安装，一般安装时间在5-10分钟！ U盘装系统 U盘装系统是目前最常用的系统安装方式。特别适合于未安装光驱的台式机或超薄笔记本电脑上。小编为了给这类无光驱用户提供最大的便利，将在本文中为大家讲解最详细的U盘装系统教程。您需要准备一个空的U盘（注意U盘中的重要数据请及时的备份出来，最好提前格式化U盘，U盘容量不小于4G）第一步：1、下载大白菜U盘制作软件到你的电脑中； 2、下载GHOST系统到你的电脑中；第二步：首先插入U盘，右键点击U盘，弹出菜单选择快速格式化（切记U盘中重要文件事先要备份出来，以防丢失），然后启动大白菜软件，界面如下图，点击界面最下方的“一键制作U盘启动”按钮，进入自动制作U盘启动盘；

用U盘重装系统教程

用U盘重装系统教程 ●装系统前的准备一个能启动电脑的U盘和一个系统的光盘镜像在安装系统前，需要准备好一些东西。一个是操作系统的镜像，另一个就是能启动的U 盘。下面我们就来讲解怎么安装deepin版的XP系统。注：读懂本文需要了解安装操作系统的一些基础知识。 ●首先是制作一个能启动电脑的带WindowsPE的启动U盘先到网上去下载一个叫“老毛桃WinPE”的工具到硬盘里，再把U盘接在电脑上，然后按下面的步骤一步步来就可以制作一个能启动的U盘了。选第4项，然后回车

输入U盘的盘符，然后回车来到格式化步骤，按默认的设置，点“开始”就行

顺利格式化

引导部分这里要说明一下，在“设备”里有两个选项，一个是电脑的硬盘，一个是要制作的U 盘。这里一定要选对U盘而别选错硬盘，从大小就能分出来哪个是U盘。笔者的U盘是2G 的，所以应该选择（hd1）[1898M]。下面的“选项”部分可以不用管，默认不勾选任何参

数就行。确认好以上步骤后，点“安装”然后进行下一步。写入引导完毕，按任意键继续要给启动U盘设个密码本来笔者不想设置启动U盘的密码，但这个步骤不能跳过，所以没办法只好设了一个密码。设置完后，一定要牢记你设好的密码，否则启动U盘会无法使用。制作完毕当你完成以上步骤后，恭喜，一个具有启动功能的U盘已经来到你的身边。你再也不用心烦没有光驱不能从光驱启动了，因为以后你可以从U盘启动再安装操作系统！想知道怎么操作吗？下一页就开始。 ●把电脑的第一启动项设为USB设备启动以往用光盘装系统，必须调整启动项为光驱启动，而现在我们要用U盘装系统，所以要调整为U盘启动。关于这个，不同电脑不同版本的bios有不同的设置方法，不过都大同小异，目的就是让电脑的第一启动项变为U盘启动。下面我们举例几个不同bios的调整方法。

超详细的电脑重装系统教程

一、准备工作: 如何重装电脑系统首先，在启动电脑的时候按住DELETE键进入BIOS，选择Advanced BIOS Features 选项，按Enter键进入设置程序。选择First Boot Device 选项，然后按键盘上的Page Up或Page Down 键将该项设置为CD-ROM，这样就可以把系统改为光盘启动。其次，退回到主菜单，保存BIOS设置。（保存方法是按下F10，然后再按Y键即可） 1.准备好Windows XP Professional 简体中文版安装光盘,并检查光驱是否支持自启动。 2.可能的情况下，在运行安装程序前用磁盘扫描程序扫描所有硬盘检查硬盘错误并进行修复，否则安装程序运行时如检查到有硬盘错误即会很麻烦。 3.用纸张记录安装文件的产品密匙(安装序列号)。 4.可能的情况下，用驱动程序备份工具(如:驱动精灵2004 V1.9 Beta.exe)将原Windows XP下的所有驱动程序备份到硬盘上(如∶F:Drive)。最好能记下主板、网卡、显卡等主要硬件的型号及生产厂家，预先下载驱动程序备用。 5.如果你想在安装过程中格式化C盘或D盘(建议安装过程中格式化C盘)，请备份C盘或D盘有用的数据。二、用光盘启动系统: (如果你已经知道方法请转到下一步),重新启动系统并把光驱设为第一启动盘,保存设置并重启。将XP安装光盘放入光驱,重新启动电脑。刚启动时，当出现如下图所示时快速按下回车键，否则不能启动XP系统光盘安装。如果你不知道具体做法请参考与这相同的-->如何进入纯DOS系统: 光盘自启动后,如无意外即可见到安装界面,将出现如下图1所示查看原图全中文提示,“要现在安装Windows XP,请按ENTER”，按回车键后,出现如下图2所示

重装系统教程

最重装系统"教程全的" 方法1: 首先，确定你的光驱是第一启动顺序并支持从光驱启动。要从光盘安装，要把BIOS进行设置，使系统能够从光盘启动。其方法如下：（1）启动计算机，当屏幕上显示 Press Del to Enter BIOS Setup 提示信息时，按下键盘上的Del键，进放主板BIOS设置界面。（2）选择 Advanced BIOS Features 选项，按Enter键进入设置程序。选择First Boot Device 选项，然后按键盘上的Page Up 或Page Down 键将该项设置为CD-ROM，这样就可以把系统改为光盘启动。（3）退回到主菜单，保存BIOS设置。（保存方法是：按下F10，然后再按Y键即可）（4）然后将光盘放入光驱，并重启电脑，系统便会从光盘进行引导，并显示安装向导界面，你可以根据提示一步步进行安装设置就OK了。在Windows XP拷贝完文件到硬盘，第一次重新启动计算机前，必须把光盘从光驱中取出，否则系统仍会从光盘启动并会循环执行安装程序。方法2: 然后，按任意键进入光驱启动模式，加载Mini版本的操作系统。然后，按Enter确定继续安装。

然后，按F8接受许可证协议。然后，选择你想要安装的位置，选择一个足够大的空间，按Enter。然后，选择文件系统，推荐使用NTFS，按Enter。然后，将进入磁盘扫描，并且将安装程序复制到硬盘上。然后，计算机将在15秒后重新启动，按Enter立即重新启动。然后，从硬盘启动继续安装过程，此时开始是图形界面模式。在进行完一系列硬件检测后，将进入区域选择提示，在此配置语言，键盘和所在地区。然后，系统将提示你输入用户名和组织名，并生成一个计算机名，你可以更改。然后，将提示输入产品注册码。然后，将提示你输入管理员密码。然后，将显示组件选择框，选择你想要安装的Windows组件。然后，将进入时区和时间的设置。然后，进入联网组件安装（如果你的计算机有网卡并在硬件检测时被正确识别）。在配置好网络之后，计算机将检测并安装一系列Windows组件及服务。然后，重新启动，运行新安装的Windows。然后，进入安装的最后阶段，监测前面没有检测到的即插即用硬件。最后，可以用了！

重装系统Win7步骤和详细教程(2014版_附系统映像)

本贴针对那些不会重装系统的用户，虽然重装系统很简单，但是还是有一些小白是不会重装的，本教程现在就详细的讲一下重装系统的具体步骤，本帖以重装Windows7系统为例，讲述一下重装系统的详细步骤。首先，重装系统之前需要将电脑中的重要资料备份和转移，这是非常重要的，备份完成后，下面我们开始重装系统第一步下载Onekey ghost下载地址 https://www.wendangku.net/doc/655995507.html,/soft/detail/23409.html 下载Windows7映像下载地址 https://www.wendangku.net/doc/655995507.html,/s/15wrIA 注意：以上两个都不要保存在C盘第二步打开Onekey ghost，选择还原分区，在GHO WIM ISO映像路径选择刚下载的Windows7.GHO。如下图

接下来，在底下选择还原到C盘（点击C盘，然后那C盘一条会变蓝）。如下图，然后点击确定。点击确定后，会弹出一个提示框（如下图），询问是否重启，点击是。接下来计算机会重启。计算机重启后，会出现下图的情况，什么都不需要做，等进度条走完即可

进度条走完后，计算机会再次重启，Windows会自动安装，什么都不需要做，等计算机进入桌面后，重装系统就算完成了。重装完成后需要做的 1.检查系统是否经过正版激活方法：控制面板--系统，拉到最下面看看是否激活（如果显示状态不可用，需要等一会）如果未激活，下载小马激活工具激活https://www.wendangku.net/doc/655995507.html,/s/1gdBUUGF 2.根据自己的使用习惯调整系统设置 3.将备份的数据导入回去 4.下载安全软件，个人推荐卡巴斯基，AVG，nod32，诺顿，小红伞，这些杀软都进入国内了，杀毒能力明显是比国产强的，这几个有的有免费版，收费的其实在淘宝几块钱就能买到一年的激活码，国产的诸如电脑管家之类的可以当做系统辅助类软件用，因为国外的杀软基本都不带清理垃圾，系统优化这之类的功能，可以两者结合使用。 5.根据自己使用习惯下载软件等等

电脑重装系统教程(XP系统重装轻松搞定)

给电脑重装系统一、准备工作: 如何重装电脑系统首先，在启动电脑的时候按住DELETE键进入BIOS，选择 Advanced BIOS Features 选项，按Enter键进入设置程序。选择First Boot Device 选项，然后按键盘上的Page Up或Page Down 键将该项设置为CD-ROM，这样就可以把系统改为光盘启动。其次，退回到主菜单，保存BIOS设置。（保存方法是按下F10，然后再按Y键即可） 1.准备好Windows XP Professional 简体中文版安装光盘,并检查光驱是否支持自启动。 2.可能的情况下，在运行安装程序前用磁盘扫描程序扫描所有硬盘检查硬盘错误并进行修复，否则安装程序运行时如检查到有硬盘错误即会很麻烦。 3.用纸张记录安装文件的产品密匙(安装序列号)。 4.可能的情况下，用驱动程序备份工具(如:驱动精灵 2004 V1.9 Beta.exe)将原Windows XP下的所有驱动程序备份到硬盘上(如∶F:Drive)。最好能记下主板、网卡、显卡等主要硬件的型号及生产厂家，预先下载驱动程序备用。 5.如果你想在安装过程中格式化C盘或D盘(建议安装过程中格式化C盘)，请备份C 盘或D盘有用的数据。二、用光盘启动系统: (如果你已经知道方法请转到下一步),重新启动系统并把光驱设为第一启动盘,保存设置并重启。将XP安装光盘放入光驱,重新启动电脑。刚启动时，当出现如下图所示时快速按下回车键，否则不能启动XP系统光盘安装。如果你不知道具体做法请参考与这相同的-->如何进入纯DOS系统: 光盘自启动后,如无意外即可见到安装界面 ,将出现如下图1所示

电脑重装系统教程

分三种情况进行介绍 1，系统能正常启动，但是运行速度慢，需要重装（这是最简单的一个） 2，系统崩溃，进不了桌面，有光驱（这个要用系统光盘） 3，系统崩溃，进不了桌面，无光驱，或者没有安装光盘的（这个用U盘装）如果不支持U盘启动又没光驱的话，那。。这个只能拆硬盘挂在别的机子上装了，好像对广大菜菜同学的挑战性还蛮高的，这里我们不介绍了。首先我们先准备一张系统盘，没有的话可以去网上下载https://www.wendangku.net/doc/655995507.html,/，下载一个系统文件（，想装WIN7的话就下载WIN7的系统，我下载的是雨林木风5周年纪念版（XP）），下载下来一般是ISO文件，然后解压出来找到里面的GHO文件，XP的话一般是600多M的，复制到E:\SYSTEM备用。下面是正文。 -------------------------------------------------------------------------------昏鸽线-------------------------------------------------------------------------------- 1，系统能正常启动，但是运行速度慢，需要重装（这是最简单的一个）这个很简单，只要下载一个小软件Onekey Ghost（下载地址：

https://www.wendangku.net/doc/655995507.html,/soft/54398.html）下载完后把软件解压出来得到的Onekey.exe复制到E:\SYSTEM，然后运行，他会自动搜索可用的系统文件，点击确定就可以了（要注意看所选的文件是否是我们下载的文件），是不是超简单的？如图 -------------------------------------------------------------------------------昏鸽线-------------------------------------------------------------------------------- 2，系统崩溃，进不了桌面，有光驱（这个要用系统光盘）首先设置光盘为第一启动项

重装系统教程(含视频)

安装windows系统教程本教程含视频教程，视频教程下载地址：https://www.wendangku.net/doc/655995507.html,/s/1qWyWKVy 在线播放地址：https://www.wendangku.net/doc/655995507.html,/show/x29A21QEI2-dvIEwk481Rw...html?from=my 当我们的电脑出现各种各样问题的时候。我们时常会很烦恼，对于新手来讲，遇到问题都不知道如何解决。其实，电脑很多问题是可以通过重装系统来解决的，按道理讲，只要电脑硬件没有损坏，都可以通过重新安装系统来解决。比如说，电脑突然变得好卡好慢，或者说无法安装应用程序，丢失什么文件啊，甚至开不了机等。最近问我装系统的人太多，所以写了此教程，让更多的人学会怎么自己安装windows 系统。只要你跟着我的步骤去做，一般都是没有问题的。特殊情况也可以直接找我帮忙解决。废话不多说，直接进入主题装系统分两种情况，一种是电脑还能开机能正常使用的，另外一种自然就是不能开机的/。下面我专门针对这两种情况做不同的教程，设备条件：一台能上网的电脑 4G及以上U盘 -------------------------------------------------------------华丽分割线--------------------------------------------------- 下面是电脑能开机情况下的安装教程，电脑无法开机的请往下下面看一、下载系统自己需要装什么系统就下载什么系统，电脑配置低点的就用32位的，高点的可以用64位的。注意32位系统最大只支持4G内存如果你电脑内存大于4G 的那就必须装64位的系统了 Windows XP下载链接：https://www.wendangku.net/doc/655995507.html,/s/1dD5y5QL密码：q61e Windows 7下载链接：https://www.wendangku.net/doc/655995507.html,/s/1wHlKi密码：rd0v Windows 8.1下载链接：https://www.wendangku.net/doc/655995507.html,/s/1eQEPWqQ密码：y6ig 上面的系统中，其中win7与win8都分32位与64位，自己选择一个下载。下面这“镜像安装器”是装系统需要用的一个工具所以必须下载下来。镜像安装器下载链接：https://www.wendangku.net/doc/655995507.html,/s/1jGh7L5s 密码：n3df 二、安装系统

用U盘装电脑系统图文教程

手把手教你能用U盘装电脑系统 ● 装系统前的准备一个能启动电脑的U盘和一个系统的光盘镜像在安装系统前，需要准备好一些东西。一个是操作系统的镜像，另一个就是能启动的U 盘。下面我们就来讲解怎么安装deepin版的XP系统。注：读懂本文需要了解安装操作系统的一些基础知识。 ● 首先是制作一个能启动电脑的带WindowsPE的启动U盘先到网上去下载一个叫“老毛桃WinPE”的工具到硬盘里，再把U盘接在电脑上，然后按下面的步骤一步步来就可以制作一个能启动的U盘了.

选第4项，然后回车输入U盘的盘符，然后回车

来到格式化步骤，按默认的设置，点“开始”就行来到格式化步骤，按默认的设置，点“开始”就行

顺利格式化这里要说明一下，在“设备”里有两个选项，一个是电脑的硬盘，一个是要制作的U盘。这里一定要选对U盘而别选错硬盘，从大小就能分出来哪个是U盘。笔者的U盘是2G的，

所以应该选择（hd1）[1898M]。下面的“选项”部分可以不用管，默认不勾选任何参数就行。确认好以上步骤后，点“安装”然后进行下一步。写入引导完毕，按任意键继续要给启动U盘设个密码本来笔者不想设置启动U盘的密码，但这个步骤不能跳过，所以没办法只好设了一个密码。设置完后，一定要牢记你设好的密码，否则启动U盘会无法使用。

当你完成以上步骤后，恭喜，一个具有启动功能的U盘已经来到你的身边。你再也不用心烦没有光驱不能从光驱启动了，因为以后你可以从U盘启动再安装操作系统！想知道怎么操作吗？下一页就开始。 ● 把电脑的第一启动项设为USB设备启动以往用光盘装系统，必须调整启动项为光驱启动，而现在我们要用U盘装系统，所以要调整为U盘启动。关于这个，不同电脑不同版本的bios有不同的设置方法，不过都大同小异，目的就是让电脑的第一启动项变为U盘启动。下面我们举例几个不同bios的调整方法。

用U盘WinPE装系统完全图文教程,教你怎样用U盘来装系统

● 装系统前的准备一个能启动电脑的U盘和一个系统的光盘镜像在安装系统前，需要准备好一些东西。一个是操作系统的镜像，另一个就是能启动的U盘。下面我们就来讲解怎么安装deepin版的XP系统。注：读懂本文需要了解安装操作系统的一些基础知识。 ● 首先是制作一个能启动电脑的带WindowsPE的启动U盘先到网上去下载一个叫“老毛桃WinPE”的工具到硬盘里，再把U盘接在电脑上，然后按下面的步骤一步步来就可以制作一个能启动的U盘了。选第4项，然后回车

输入U盘的盘符，然后回车来到格式化步骤，按默认的设置，点“开始”就行

顺利格式化引导部分

这里要说明一下，在“设备”里有两个选项，一个是电脑的硬盘，一个是要制作的U盘。这里一定要选对U盘而别选错硬盘，从大小就能分出来哪个是U盘。笔者的U盘是2G的，所以应该选择（hd1）[1898M]。下面的“选项”部分可以不用管，默认不勾选任何参数就行。确认好以上步骤后，点“安装”然后进行下一步。写入引导完毕，按任意键继续

要给启动U盘设个密码本来笔者不想设置启动U盘的密码，但这个步骤不能跳过，所以没办法只好设了一个密码。设置完后，一定要牢记你设好的密码，否则启动U盘会无法使用。制作完毕

联想U410重装系统win8启用intel硬盘加速超详细教程

联想U310/U410重装系统win7\win8启用intel硬盘加速超详细教程前期准备工具： 1.制作U盘启动映像文件，具体制作教程在这里： https://www.wendangku.net/doc/655995507.html,/thread-3189-1-1.html 2.下载Intel Rapid Storage Technology 驱动，下载完毕后拷贝到U盘下载地址： https://www.wendangku.net/doc/655995507.html,/thread-3188-1-1.html 3.开机Fn+F2进入bios，设置boot-UEFI Boot --disabled，如果不设置安装系统的时候会提示无法再将系统安装到硬盘，因为此硬盘有mbr表，设置完毕后Fn+F10保存退出。相机型号：MiOne 曝光时间：光圈： 4.安装操作系统之前请保证以下表选项是开启的选择Configuration> SATA controller working mode，改为“RAID”；选择Configuration>IntelRapid Start Technology, 改iRST Support和Entry on S3 critical battery event两个都为“Enable”；选择Configuration>MemoryRemap 为“Enabled”； 5.去联想官网下载您的驱动，等系统安装完毕后好打驱动。开始安装操作系统

1.开机时按Fn+F12，进入启动盘选这项，这里当然要选择我们的U盘，进入安装相机型号：MiOne 曝光时间：光圈：

2.到这里会发现看不到硬盘，我们点击加载驱动程序相机型号：MiOne 曝光时间：光圈：

电脑重装系统详细图文教程

电脑重装系统一、前言电脑现在是日常生活必备品，不过我们在新买电脑和电脑出现各种问题时都会遇到装系统的问题，而重装系统也就成为了必备技能，但是很多朋友可能是电脑的初级使用者，不知道如何给电脑做系统。其实装系统是很简单的。下面教大家如何重装系统。首先要说明的是两种装系统的方法： 1、系统盘重装（一般买电脑的地方都有系统盘买） 2、U盘重装系统（现阶段最常用的）在这里我主要讲解如何用U盘装系统二、U盘装系统 1、工具 U盘、电脑系统软件、U盘启动盘制作工具。当然你还需要一台电脑

2、电脑软件下载地址推荐需要啥样的系统下载啥系统XP win7 32/64 win8等等。https://www.wendangku.net/doc/655995507.html,/ https://www.wendangku.net/doc/655995507.html,/

上面的是网站，也可以去百度贴吧，论坛等等地方找。 3、U盘启动盘制作工具一键工作室、大白菜、小马PE、U盘装系统，等等。非常多喜欢哪个下哪个。三、详细步骤 1、工具制作下载U盘启动盘制作工具（以一键工作室为例，其他软件稍有区别但本质一样，一通就可以万通）

下载好之后安装，然后打开软件并在电脑上插入你要做成启动盘的U盘。如下图点击一键制作USB启动盘。等待片刻制作好就行了。如下图

2、将你下载好的系统软件转入制作好的U盘当中

将下载好的系统软件压缩包，解压之后将后缀名为GHO文件放进制作好的U盘里，这样我们就可以开始做系统了。 3、装系统 1）在要做系统的电脑上插上优盘 2）开机按F12选择USB启动（笔记本电脑），这里是按完开关按钮之后直接按F12。注：台式机和一些个别的笔记本电脑需要进入BIOS界面更改，大家可以百度一下进入BIOS的方法和更改设置的方法，以后我也会对这一方面详细总结一下。 3）启动并进入界面（如下图

重装系统傻瓜教程

自己有电脑，而且不会自己装系统的啊，每次系统坏了都要花钱找别人弄的啊！赶紧过学习下，电脑都用这么久了，还不会做这些，别人会笑话的！！哈哈哈。。。开始介绍吧！！给电脑装系统一般有4种办法： 1. 傻瓜式装系统：可装Win7或WinXP，主要的操作如下：需要准备的东西：现有的电脑操作系统，（例如你的电脑系统还能用，但是很不给力！你想重装一下系统，或者换个系统等情况）；到网上去下载一个GHO格式的镜像文件的系统；（百度一搜一大堆！记住你下载是什么类型的系统，就能装什么类型的系统，比如WinXP，Win7，或者64位的系统）；还有一个最重要的东西，那就是Ghost备份还原工具，是EXE格式的应用程序；（百度搜“Ghost 备份还原工具”，就有）具体如下图好！准备工作好了，下面开始装系统，第一步：打开Ghost备份还原工具，界面如下:

看好！第一行选还原系统，第二行选择你下载那个GHO格式的系统【特此声明，如果你发现你选择好的GHO文件后，然后下面选择C盘后发现最底下的“确认”按钮还是灰色不能点击；则说明你下载的系统是错误的GHO和格式的系统，建议你去重新下载GHO格式的系统，当然上图的XP系统是假的，我用压缩包改造的！】如果选中C盘后，那个确认按钮可以使用，就点击那个确认按钮，它会弹出一个对话框；问你真的要重新启动还原系统吗？你点击是，它会自动重启并为你重新安装系统，好了，重启时你就不用动电脑了，你可以一边玩去了，他是全自动的安装系统；过个几分钟就能装好系统了！这就是最简单的傻瓜式装系统！这样装系统的缺点就是：你装的系统是被别人搞过的！不是完全的纯净版，或者微软的原版系统，这种系统里面会自带一些你不喜欢的软件，或者说你根本用不到的软件！优点除了系统自己可以装好部分驱动，其他的我就不说了，你懂的！ 2.可移动磁盘（USB Device）需要准备东西：能用的可移动磁盘（U盘，TF卡，SD卡，MP3等等随便，其中内存至少大于512MB）；WindowsPE（ISO格式的镜像文件，可在网上搜索下载，文件大小在100MB~300MB左右，太大的不好用，启动慢）； WindowsXP或Windows7（是你要安装在电脑上的系统，最好是ISO格式镜像文件，WinXP大约600MB左右，Win7 32位大约2.5GB，64位的大约3.2GB左右

教你怎么制作系统GHOST封装详细图文教程完整版

xp系统GHOST封装详细图文教程完整版（适合初学者）教程架构：第一篇系统、工具及软件安装第二篇封装工具选择及实战第三篇光盘ISO文件制作下面以封装制作GHOSTXPSP3为例，进行讲解！第一篇系统、工具及软件安装一、准备工作 1、操作系统选择：建议采用微软官方的VOL原版 ——为什么要用VOL原版因为VOL原版适用于任何电脑，而某些品牌机赠送的是OEM版，只能用于对应的品牌电脑，并且还需激活！ ——特别说明一下：很多人喜欢说正版，其实所谓的正版是要通过微软官方验证的，但是系统内容却并不一定是原版的。 Windows XP with sp3 VOL 微软原版下载：

SHA1: D142469D0C3953D8E4A6A490A58052EF52837F0F CRC32: FFFFFFFF ？正版密钥： MRX3F-47B9T-2487J-KWKMF-RPWBY M6TF9-8XQ2M-YQK9F-7TBB2-XGG88 附：关于VOL 和 VLK 解析—— VOL是Volume Licensing for Organizations 的简称，中文即“团体批量许可证”。根据这个许可，当企业或者政府需要大量购买微软操作系统时可以获得优惠。这种产品的光盘卷标带有"VOL"字样，就取 "Volume"前3个字母，以表明是批量。这种版本根据购买数量等又细分为“开放式许可证”(Open License)、“选择式许可证(Select License)”、“企业协议(Enterprise Agreement)”、“学术教育许可证(Academic Volume Licens ing)”等5种版本。根据VOL计划规定， VOL产品是不需要激活的。 VLK不是一种独立版本。VLK的全称是Volume Licensing Key ，它是指VOL版本在部署(deploy)中所需要的 KEY，而且只能用于VOL版中。简而言之，对VOL版XP来说，VLK就是PID为640的安装 KEY 。例如，上海政府VOL版XP所带的安装KEY：DG8FV-B9TKY-FRT9J-6CRCC-XPQ4G，就是一个VLK。VLK的作用仅仅是证明产品合法化。按照许可协议，零售版XP的“密钥”只能用在一台PC上，而 VOL 版XP 则可按协议供给多台PC使用。还有一个重要的区别，零售版XP需要激活，而 VOL 版XP根本就没有这个概念。VOL版的XP没有Home这个版本，其功能跟 Professional版是一样的。 2、系统补丁：主要靠自己平时收集整理，如果没有，可以使用别人做好的，也可以使用360安全卫士下载，然后收集整理。 3、办公软件：一般来讲，做GHOST封装都会安装OFFICE办公软件，也建议采用微软原版，不要使用修改版。

重装系统四种简单方法

给电脑装系统一般有4种办法： 1. 傻瓜式装系统：可装Win7或WinXP，主要的操作如下：需要准备的东西：现有的电脑操作系统，（例如你的电脑系统还能用，但是很不给力！你想重装一下系统，或者换个系统等情况）；到网上去下载一个GHO格式的镜像文件的系统；（百度一搜一大堆！记住你下载是什么类型的系统，就能装什么类型的系统，比如WinXP，Win7，或者64位的系统）；还有一个最重要的东西，那就是Ghost备份还原工具，是EXE格式的应用程序；（百度搜“Ghost备份还原工具”，就有）具体如下图

好！准备工作好了，下面开始装系统，第一步：打开Ghost备份还原工具，界面如下:

看好！第一行选还原系统，第二行选择你下载那个GHO格式的系统【特此声明，如果你发现你选择好的GHO文件后，然后下面选择C盘后发现最底下的“确认”按钮还是灰色不能点击；则说明你下载的系统是错误的GHO和格式的系统，建议你去重新下载GHO格式的系统，当然上图的XP系统是假的，我用压缩包改造的！】如果选中C盘后，那个确认按钮可以使用，就点击那个确认按钮，它会弹出一个对话框；问你真的要重新启动还原系统吗？你点击是，它会自动重启并为你重新安装系统，好了，重启时你就不用动电脑了，你可以一边玩去了，他是全自动的安装系统；过个几分钟就能装好系统了！这就是最简单的傻瓜式装系统！这样装系统的缺点就是：你装的系统是被别人搞过的！不是完全的纯净版，或者微软的原版系统，这种系统里面会自带一些你不喜欢的软件，或者说你根本用不到的软件！优点除了系统自己可以装好部分驱动，其他的我就不说了，你懂的！ 2.可移动磁盘（USB Device）需要准备东西：能用的可移动磁盘（U盘，TF卡，SD卡，MP3等等随便，其中内存至少大于512MB）； WindowsPE（ISO格式的镜像文件，可在网上搜索下载，文件大小在100MB~300MB左右，太大的不好用，启动慢）； WindowsXP或Windows7（是你要安装在电脑上的系统，最好是ISO

重装系统Win7步骤和详细教程 U盘安装(2015版)

安装电脑系统已经两年多，主要是品牌笔记本与台式机，戴尔，三星，华硕等。我发现其实很多人不会安装现在的新的品牌电脑，包括一些电脑店的，有些打酱油的电脑店，因为品牌机器和组装机器安装有些区别，所以有的人搞不定了就对客户糊弄过去，这样的机器客户用起来其实问题多多，要么驱动没有装好，例如ATI的6370，如果用万能驱动很容易打驱动打成6300.这个是错误的,游戏或者看高清容易卡机出现蓝屏现象，最好到官网下载官方显卡驱动。有的人系统版本装的不对也容易出问题，现在的笔记本都不建议装xp，都建议装win7，这样兼容性会好点。废话少说，开始教学装系统了：······ 首先：准备个U盘，U盘最少8G 因为系统要占据差不多4G 其次：下载PE软件，简称U盘PE吧。第三：下载好PE后那就是制作PE系统制作步骤如下：一、制作前准备（注意：操作前备份好u盘数据-因为U盘会格式化） 1.电脑内存不能小于512MB 2.U盘的容量大于512MB，建议8G 3.下载老毛桃U盘启动盘制作工具，如果已经下载好久跳过下载下载地址：https://www.wendangku.net/doc/655995507.html, 二、解压下载的老毛桃U盘启动制作工具，双击【老毛桃U盘启动盘制作工具 Build20120501】,选择你的U盘，画面如下图：

点击“一键制成USB启动盘”按钮(注意操作前备份重要数据)

制作成功，如下图，此时可以拔出你的U盘注意：由于U盘系统文件隐藏，你会发现u盘空间会减少330M左右，请不要担心此时没有制作成功三、将系统文件复制到U盘这里说的系统文件是XP镜像或者win7镜像系统，这里都把下载地址给大家，我这个win7系统已经安装了几千台电脑的了，所以稳定性是不用说的，下载地址： win7系统下载地址下载地址2：https://www.wendangku.net/doc/655995507.html,/xitong.html 下载地址1：https://www.wendangku.net/doc/655995507.html,/share/home?uk=3476242497

电脑重装系统的详细步骤

电脑重装系统的详细步骤一、准备工作: 如何重装电脑系统首先，在启动电脑的时候按住DELETE键进入BIOS，选择 Advanced BIOS Features 选项，按Enter键进入设置程序。选择First Boot Device 选项，然后按键盘上的Page Up或Page Down 键将该项设置为CD-ROM，这样就可以把系统改为光盘启动。其次，退回到主菜单，保存BIOS设置。（保存方法是按下F10，然后再按Y键即可） 1.准备好Windows XP Professional 简体中文版安装光盘,并检查光驱是否支持自启动。 2.可能的情况下，在运行安装程序前用磁盘扫描程序扫描所有硬盘检查硬盘错误并进行修复，否则安装程序运行时如检查到有硬盘错误即会很麻烦。 3.用纸张记录安装文件的产品密匙(安装序列号)。 4.可能的情况下，用驱动程序备份工具(如:驱动精灵 2004 V1.9 Beta.exe)将原Windows XP下的所有驱动程序备份到硬盘上(如∶F:Drive)。最好能记下主板、网卡、显卡等主要硬件的型号及生产厂家，预先下载驱动程序备用。 5.如果你想在安装过程中格式化C盘或D盘(建议安装过程中格式化C盘)，请备份C盘或D盘有用的数据。二、用光盘启动系统: (如果你已经知道方法请转到下一步),重新启动系统并把光驱设为第一启动盘,保存设置并重启。将XP安装光盘放入光驱,重新启动电脑。刚启动时，当出现如下图所示时快速按下回车键，否则不能启动XP系统光盘安装。如果你不知道具体做法请参考与这相同的-->如何进入纯DOS系统: 光盘自启动后,如无意外即可见到安装界面 ,将出现如下图1所示

重装系统步骤-小白一键重装系统图文教程

时下电脑已经化作普通电子商品进入千家万户，然后大家在使用的过程中经常会遇到的一个问题就是如何来重装电脑的系统，即使是对于对电脑比较熟悉的用户，也未免知道系统安装的几种方式，怎么重装系统是最快最好的。下面请带着这些问题，让我为大家一一讲解。本文针对需要重装系统的用户而编写的简易化图文教程，旨在让广大对系统安装技术空白的电脑使用者普及这一系统的维护技巧！首先看到我写的标题请勿喷，看完你就发现自己一分钟就掌握电脑装系统的技巧！下面介绍给大家的前所未有的简单装机方法要点是-------只需看完以下的步骤 1、打开小白一键重装系统软件（免安装，仅7.88M,由于论坛不支持上传超过5M的，所以就不提供了），点击打开“小白一键重装系统”得到下图所示软件界面，1、选择你想要安装的系统目标路径，点击选项“安装到此分区”，下一步（一般默认选择C盘即可，安装前请对该磁盘做好数据备份）第二步、然后的话就是选择你所需要的系统，大家根据自己的电脑的性能再结合个人的使用习惯选择适合你的系统即可，软件自带xp/win7的32位以及64位系统，足以满足各位同学的不同需求的了，选定好的话点击“安装此系统”，再下一步

第三步、软件进行一个自动下载相关组件的过程，界面上是一段进度条的过程，大家稍等些许时间即可，下载完毕点击提示框的“是”重启。

第四步、这是重启后的系统进行安装还原的一个界面，相信很多同学也见过的了，对此应该不陌生，系统还原进度结束后是蓝色界面的驱动安装阶段，以上都是全自动的，无需你任何操作，非常人性化的，因为这个软件就是专门为普通的电脑用户设计，就连女生都可以自己来操作完成的，可以说是最棒的装机神器！

电脑重装系统详细步骤

当电脑遇到问题怎么办？解决90%与100%问题的重启、重买相信大家都会，有钱钱都不是问题的啦~那没有钱钱怎么办？没错，接下来要介绍的就是解决99%问题的重装系统方法！重装系统其实就是重新安装电脑的操作系统，电脑问题一般分为两类：一类是硬件问题，这个就要更改硬件；另一类是软件方面的问题，包括系统出错、崩溃、中毒等。而重装系统解决的就是软件方面的问题。这里介绍两种重装方法：一、U盘重装系统方法需要用到的工具： ●小白一键重装系统软件 ●一个U盘

1、首先需要用软件将U盘制作成启动盘，插入U盘然后打开小白一键重装系统（制作启动盘会格式化U盘，U盘有东西需要提前做好备份） 2、打开制作系统选项（制作启动盘），选择好U盘后按默认参数点击开始制作。 3、选择一个系统镜像勾选点击制作，然后等待制作完成，这样U盘启动盘就制作成功

了。 4、将U盘启动盘插在需要重装系统的电脑上，重启电脑开机出现第一个画面时按下U 盘启动快捷键，快捷键在电脑开机的第一个画面有短暂的提示，如下图：也可以根据自己的电脑型号查询：

5、按下快捷键后会调出快捷启动菜单，按方向键与回车键选择自己的U盘名字进入到 PE选择界面。

6、P E选择界面一般选择新机型的PE，如果新机型的PE不能正常工作那么选备用的旧机型。 7、进入PE后打开装机工具，勾选自己下载的系统镜像文件，选择需要安装的分区（也就是系统盘，一般是C盘，进入PE后盘符可能会改变，可以打开计算机查看自己的系统在哪个盘，然后选择这个盘），点击开始安装。

8、上面的那一步后可以说已经成功一半了，后面的步骤基本是自动完成的，等待一段时间安装完成后重启电脑。 9、之后就可以看到熟悉的系统启动画面了，这是新系统的第一次开机，会进行多次配