Patent Application: US-201515311590-A

Abstract:
provided are a platelet - derived growth factor b derivative , the encoding nucleic acid molecule thereof , and a vector and host cell having the nucleic acid molecule . also provided are a preparation method for the mutant , and the use of the mutant in preparing medications for promoting cell division , cell proliferation , wound healing , skin regeneration , bone and tooth defect regeneration , and joint repair .

Description:
the embodiments of the present invention will be described in detail below in combination with the examples , however , those skilled in the art will appreciate that the following examples are merely intended to illustrate the invention and should not be construed as limiting the scope of the invention . those without the specific conditions specified in the examples should be carried out under normal conditions or the conditions recommended by the manufacturer . the reagents or instruments without manufacturers specified are all commercially available conventional products . in previous studies , we have successfully employed pichia pastoris expression system to express rhpdgf - bb thr6 with five amino acids deleted at n - terminus with a expression level of up to 100 mg / l ( see cn patent no . : zl200410068993 . 2 ). pdgf - b thr6 is selected as research subject in order to ensure homogeneity of expressed protein without biological activity impaired . however , further studies demonstrate that , rhpdgf - b thr6 monomer expressed by pichia pastoris still exhibits various forms with heterogeneous molecular weights ranging from 10 to 15 kda ( fig2 ). the following examples are carried out by engineering based on rhpdgf - b thr6 , and all the descriptions of the sites or positions are based on the wild - type pdgf - b ( 109 amino acids ). genbank number of the amino acid sequence of the wild - type pdgf - b is nm - 002608 . 2 . dna sequences encoding various pdgf mutants were synthesized by shanghai sangon inc . the gene fragments were cloned into the expression vector pmex9k ( see patent zl02117906 . 9 ) via restriction sites xhoi and ecori and confirmed by sequencing . the recombinant plasmids were extracted , linearized by sali digestion , and then transformed into pichia pastoris expression strain gs115 competent cells by electroporation . the yeast transformants were screened by histidine - deficient md plates and the positive recombinant yeast strains were identified by pcr . the single clones of the recombinant yeast strain were inoculated into a flask of 25 ml bmgy medium ( bmgy medium is prepared as follows : 10 g yeast extract powder and 20 g tryptone were weighed , dissolved in 700 ml water , autoclaved at 121 ° c . for 20 min ; cooled to room temperature , added 100 ml 1 m potassium phosphate buffer , 100 ml 10 × ynb and 100 ml 10 × gy , and stored at 4 ° c . wherein : 10 × ynb ( 13 . 4 % yeast nitrogen source base ), 10 × gy ( 10 % glycerol ), 1 m potassium phosphate buffer ( 132 ml 1m k 2 hpo 4 and 868 ml 1m kh 2 po 4 were measured , adjusted to ph 6 . 0 ± 0 . 1 with phosphoric acid or koh , autoclaved at 121 ° c . for 30 min and stored at room temperature .) yeast extract ( lp0021 ) is a product of oxoid inc . and peptone ( 211677 ) is a product of b & amp ; d inc . ), cultured and propagated at 28 - 30 ° c . with 220 - 250 rpm to od 600 = 2 - 6 ( about 16 - 18 hours ). 25 ml yeast culture was inoculated into a flask containing 1 l bmgy , and continued to culture and propagate at 28 - 30 ° c . with 220 - 250 rpm to od 600 = 2 - 6 . yeasts were collected by centrifugation at room temperature with 1500 - 3000 g for 5 min . the supernatant was removed and the yeasts were resuspended with 1 l bmmy medium to initiate expression induction . the induction temperature was 28 ° c . and the rotational speed was 220 rpm . methanol was added every 24 hours until the final concentration is 0 . 5 %, and the induction time was 72 hours . after the induction was complete , the supernatant containing the recombinant protein was collected by centrifugation with 7000 rpm at room temperature . the expression supernatant of pichia pastoris was adjusted into an appropriate buffer by centrifugation and filtration , and subsequently subjected to hydrophobic interaction chromatography ( phenyl sepharose 6 fast flow ), ion exchange chromatography ( source 30s ) and gel filtration chromatography ( hiload superdex 75 prep grade ) to obtain the protein of interest with a purity & gt ; 95 % ( fig5 ). the chromatographic media are all products from ge amersham bioscience inc . hydrophobic chromatography was carried out as follows . ( 1 ) yeast expression supernatant was adjusted for conductivity with ½ volume of conditioning buffer ( 60 mm pb , 3m ( nh 4 ) 2 so 4 , ph 7 . 2 ). ( 2 ) as described in the instruction , the column was equilibrated with an equilibration buffer ( 20 mm pb , 1m ( nh 4 ) 2 so 4 , ph 7 . 2 ). ( 3 ) the sample was loaded to the column , thereafter the column was washed with the equilibration buffer until the baseline is flat . ( 4 ) the column was eluted with an elution buffer ( 20 mm pb , 50 % ethylene glycol , ph 7 . 2 ) to collect the protein of interest . ion exchange chromatography was carried out as follows . ( 1 ) the phenyl hs elution peak was diluted with an equilibration buffer ( 20 mm pb , ph 7 . 2 ) to a conductivity of 6 ms / cm or less . ( 2 ) according to the method in the instruction , the column was equilibrated with the equilibration buffer . ( 3 ) the sample was loaded to the column , thereafter the column was washed with the equilibration buffer until the baseline is flat . ( 4 ) the column was eluted with a gradient of elution buffer ( 20 mm pb , 1m nacl , ph 7 . 2 ) to collect the protein of interest . gel filtration chromatography was carried out as follows . ( 1 ) the column was equilibrated with pbs buffer ( 20 mm pb , 0 . 15m nacl , ph 7 . 2 ). ( 2 ) the source 30s elution peak was loaded with a loop , and the volume of each loading was not more than 3 % of the column volume . ( 3 ) the column was washed with pbs buffer to collect the protein of interest . 30 μl purified protein with a suitable concentration was added to 10 μl 4 × sds - page buffer ( with and without 20 mm dtt ) respectively , denatured at 100 ° c . for 5 min and centrifuged . then 30 μl of the supernatant was taken for sds - page electrophoresis analysis ( separation gel is 15 %). following the electrophoresis , the gel was stained with coomassie brilliant blue r250 . the sample was prepared in the same way as in sds - page . 3 μl sample was taken for sds - page . following the electrophoresis , the proteins were transferred to a nitrocellulose membrane with 300 ma constant current for 1 h and blocked with 5 % skim milk / tbst at room temperature for 1 h . the primary antibody pdgf - b ( f - 3 ) ( santa cruz biotechnology , sc - 365805 ) was 1 : 1000 diluted , coated at room temperature for 1 h , and washed with tbst for several times . hrp - labeled secondary antibody ( cell signaling technology , # 7076 ) was 1 : 10000 diluted , incubated at room temperature for 1 h , and washed with tbst . the substrate was added and imaged with an las400 mini gel imaging system ( ge ). sample preparation and sds - page processes were the same as the above . following the completion of the electrophoresis , the proteins were transferred to a pvdf membrane with caps electroblotting buffer at 300 ma constant current for 1 h , and stained with 0 . 1 % coomassie brilliant blue r250 , immediately after that , fully decolored with 50 % methanol until the protein bands were visible . the protein bands to be determined were cut off and sent to chromatography laboratory of biomedical analysis center , military medical academy for determination . 5 μl of the sample and the positive control ifn - co were added into 3 μl of 10 × glycoprotein denaturation buffer ( containing neb pngase f enzyme ) and 15 μl of water , and heated at 100 ° c . to denature for 10 min . after cooling , 3 μl np - 40 , 3 μl g7 buffer ( containing neb pngase f enzyme ) and 2 μl peptide n - glycosidase f ( pngase f ) ( a product of new england biotech inc . ( neb )) were added and digested at 37 ° c . for 3 h . following the completion of the digestion , the sample was heated at 100 ° c . to inactivate the enzyme and then subjected to sds - page electrophoresis . following the completion of the electrophoresis , staining was performed using a glycoprotein staining kit ( themo scientific , # 24562 ). firstly , the gel was added into 100 ml 50 % methanol and fixed for 30 min ; the gel was washed several times with 3 % acetic acid , transferred to 25 ml oxidizing solution and shaken gently for 15 min ; the gel was washed several times with 3 % acetic acid , transferred to 25 ml glycoprotein staining reagent and shaken gently for 15 min ; thereafter , the gel was transferred to 25 ml reducing solution and shaken gently for 5 min ; then , the gel was washed with 3 % acetic acid and rinsed with deionized water . balb / c 3t3 cells ( purchased from beijing xiehe cell resource center ) were cultured in dmem complete medium ( life technology ) containing 10 % fbs under a condition of 37 ° c . and 5 % carbon dioxide . after digestion and collection , the cells were prepared as cell suspension containing 5 . 0 × 10 4 cells per ml with a complete broth , inoculated into a 96 - well cell culture plate ( 100 μl per well ), and followed by culturing under a condition of 37 ° c . and 5 % carbon dioxide . 24 hours later , the medium was exchange into a maintenance medium ( dmem containing 0 . 4 % fbs ), followed by culturing under a condition of 37 ° c . and 5 % carbon dioxide . upon 24 hours of culturing , the culture medium was discarded and added a pre - gradient diluted pdgf - bb solution ( 100 μl per well ). the cells were cultured for another 64 to 72 hours under the action of proteins , and were assayed for cell proliferation with wst - 1 method as follows : 10 μl wst - 1 solution ( roche , 11644807001 ) was added into each well , cultured under a condition of 37 ° c . and 5 % carbon dioxide for 3 hours , and then measured for the absorbance at a wavelength of 450 nm using a microplate reader ( reference wavelength : 630 nm ). the experimental data were processed by a four - parameter regressive calculation method . the ec 50 values of the two proteins were calculated respectively . the experiment was repeated three times . the difference statistical analysis between the two , groups was performed by t - test . example 1 . the co - effect of the proteolysis and glycosylation contributes to the formation of diverse monomers of pdgf - bb thr6 the inventors firstly suspected that proteolysis is the cause of the formation of diverse pdgf - b monomers . by reducing sds - page electrophoresis , different monomers of pdgf - b were separated and five bands were detected via coomassie brilliant blue staining ( fig3 a ). the five protein bands were subjected to sequencing for n - terminal amino acid sequence , and the results showed that the first five amino acid residues at n - terminus in the first , second and third bands were all tiaep , corresponding to the correct n - terminal sequence of pdgf - b thr6 , while the first five amino acid residues at n - terminus in the fourth and fifth bands were tnanf . the alignment of protein sequences determined that the fourth and fifth protein fragments were the truncated proteins generated by proteolytic cleavage at arg32 - thr33 ( fig3 a ). however , this cannot explain the reason for the formation of at least 5 kinds of pdgf monomers . the difference in molecular weights between bands 1 , 2 , 3 and bands 4 , 5 might be due to c - terminal cleavage . to answer this question , the inventors performed wb assay using a specific monoclonal antibody ( f - 3 ) against pdgf - b c - terminus ( santa cruz biotechnology , sc - 365805 ). the result showed that only two of the five bands were detected . but interestingly , the two bands bound to the antibody appeared to correspond to the third and fifth protein fragments ( fig3 b ). if the first , second , and fourth protein fragments cannot be detected by the antibodies due to the c - terminal cleavage , their molecular weights should be smaller . however , this is clearly not consistent with the result of electrophoresis assay . this means that there are other reasons to be found . in order to analyze whether pdgf - b was glycosylated , pdgf was digested with peptide n - glycosidase f ( pngase f ), and sds - page and glycoprotein staining were performed simultaneously . pngasef is an amidase which can act on almost all n - glycan chains in a glycopeptide / glycoprotein , cleaves between the innermost glcnac and asparagine residues of the sugar chain moiety , and converts asparagine into aspartic acid ( 10 ), and is the most widely used enzyme in the identification of n - glycoprotein in the glycoprotein proteomics research . the recombinant ifn - w protein expressed in pichia pastoris acts as a positive control of n - glycosylated protein . coomassie brilliant blue staining result showed that there was no change in the relative molecular weight of pdgf - b protein before and after the cleavage , indicating that pdgf - b protein did not undergo n - glycosylation ( fig3 c , left ). however , the glycoprotein staining result indicated that pdgf - b is indeed a glycoprotein ( fig3 c , right ). this means that pdgf - b secreted by pichia pastoris was o - glycosylated . meanwhile , further analysis showed that only three protein fragments were detected in the sugar staining , which should correspond to bands 1 , 2 and 4 in the sds - page result respectively ( fig3 b ). this is also consistent with the result of the above wb assay : the first , second and fourth protein fragments were glycosylated at c - terminus thereof , thus affecting the binding of pdgf - b thr6 to the antibody . combining the above experimental results , the inventors deduced that the several forms of pdgf - b thr6 monomers resulted from the co - effect of the proteolysis and differentially post - translational glycosylation occurring between amino acids arg32 - thr33 at positions 27 / 28 ( table 1 ). moreover , the inventors would like to confirm the above - mentioned deduction , and expected the expression of pdgf - b in pichia pastoris to be homogenous . firstly , the inventors would like to determine the possible o - glycosylation sites . the prediction of the glycosylation sites of pdgf - b thr6 protein sequence was performed using online website cbs ( www . cbs . dtu . dk ) ( 11 ). the result showed that thrs at positions 6 , 101 and 109 are the possible o - glycosylation modification sites ( fig4 ). compared with n - glycosylation , there is no definite motif for o - glycosylation sites , so the prediction thereof is also relatively difficult . however , the predicted potential glycosylation sites at positions 6 , 101 and 109 are consistent with our results : there should be glycosylation modifications ( thr101 , thr109 ) at c - terminus of the pdgf - b thr6 protein , since they hindered the binding to the antibody ; there should be glycosylation modification site ( s ) before thr33 , which could explain the fact that there was only one ( band 4 ) glycosylation - modified variant for the digested pdgf - b , but there were two bands ( bands 1 , 2 ) for glycosylated pdgf - b monomer without digestion ( fig3 b ; table 1 ). in order to confirm the predicted results , we constructed two mutants pdgf - m1 and pdgf - m2 of pdgf - b thr6 . two glycosylation sites at c - terminus were mutated in pdgf - m1 , and all three potential glycosylation sites were mutated in pdgf - m2 ( see fig6 a for the pattern of mutations ). meanwhile , in order to remove the protease cleavage site arg32 - thr33 , we mutated arg32 to pro , considering the evolutionary selection of amino acids . we noted that the mature pdgf - b protein has 60 % amino acid sequence homology with pdgf - a , and both have a high similarity in terms of structure and function , whereas the amino acid in the corresponding position of pdgf - a protein is pro . dna sequences encoding pdgf - m1 and pdgf - m2 were inserted into expression vector pmex9k , and integrated into the pichia pastoris strain gs115 . the expression was induced by methanol and proteins were purified via chromatography . the purified pdgf - b proteins were subjected to sds - page , glycoprotein staining and western blotting detection , in order to determine the properties of the engineered protein . wb result showed that only single band could be detected after the two mutants were reduced , and the relative molecular mass is about 12 kda , consistent with the expected one ( fig6 b ). sds - page result showed that pdgf - m2 is a single band , but pdgf - m1 still has a minor band above the major band ( fig6 c ). it is presumed that this band results from the glycosylation of thr6 . glycoprotein staining result showed that the glycosylation levels of the two mutants were very low compared to those before mutation and hardly to be detected with glycoprotein staining ( fig6 d ). the above results indicated that mutation of r at position 32 to p removed the potential kex2 protease cleavage site and prevented the formation of thr33 truncated pdgf - b monomer , while the mutations of three glycosylation sites thr6 , 101 and 109 also abolishes the post - translational glycosylation modifications of the protein at different degrees , thereby rendering the expression of pdgf - b protein in pichia pastoris homogenous . in order to analyze whether the mutations of glycosylation sites thr6 - ala , thr101 - ala and thr109 - ala and the mutation of arg32 - pro kex cleavage site could affect the biological activity of pdgf - bb , the inventors determine the proliferation activity of pdgf - b thr6 and pdgf - m2 on balb / c 3t3 cells using wst - 1 method . the results showed that ec50 of pdgf - b thr6 is 5 . 434 ± 0 . 6475 ng / ml , while ec50 of pdgf - m2 is 3 . 492 ± 0 . 4078 ng / ml . the t - test showed that the protein activity after engineering is higher than before engineering , with p value of 0 . 0117 ( fig7 ). in order to enhance the expression level of pdgf - m2 , the inventors carried out codon - optimization on pdgf - m2 ( pdgf - im - p ) according to the codon preference of pichia pastoris during protein expression using online tool java condon adaptation tool . the optimized encoding dna sequence is as follows : on the basis of codon optimization , arg32 was mutated to val ( pdgf - im - v , val codon used is gtt ) and ile ( pdgf - im - i , ile codon used is atc ), the expression level was compared with that of pdgf - m2 in order to analyze the effect of mutation of arg32 on protein expression . the dna sequences encoding pdgf - im - p , pdgf - im - v and pdgf - im - i were ligated to the sequences such as restriction site ( s ), terminator ( s ), cloned into expression vector pmex9k , and integrated into expression strain gs115 . following histidine - deficient md plate screening , nine clones were randomly selected , and subjected to expression induced by methanol in a tube . the sds - page electrophoresis analysis of the culture supernatant demonstrated that the expression amount of pdgf - im - p protein was significantly higher than other two strains ( fig8 a ). the screening of gs115 / pdgf - im - p , gs115 / pdgf - im - v , and gs115 / pdgf - im - p clones with multiple inserts was carried out with g418 . the expression of clones grown on plates with 2 . 0 mg / ml and 4 . 0 mg / ml g418 was analyzed , respectively . the result demonstrated that the average expression level of pdgf - im - p was higher than the other two strains ( fig8 b ). this indicates that the mutation of arg32 site would affect the secretion and expression level of pdgf in pichia pastoris , and the mutation of this site to pro is relatively favorable for expression . example 5 lc / ms detection of the glycosylation of pdgf - b and pdgf - m2 mutants the recombinant pdgf - b wild - type and pdgf - m2 mutants were reduced with dtt ( 2 . 5 mm ) at 37 ° c . for 30 min , and diluted with buffer a ( an aqueous solution containing 0 . 1 % formic acid ), followed by liquid chromatography and mass spectrometry ( lc / ms ) analysis . the proteins were separated on easy - spray column ( 15 cm × 75 m id , 3 - μm c18 particles ) using easy - nlc system ( thermo fisher scientific ), eluted with a linear gradient of buffer b ( containing a solution of 0 . 1 % formic acid in methanol ; 0 - 90 %, 20 min ) at a flow rate of 300 nl / min . high resolution spectra were obtained using q exactive mass spectrometer ( thermo fisher scientific ) under a condition of a resolution of 60 , 000 , m / z 350 - 1600 and de - convoluted using xtract software ( thermo scientific ). analysis of the pdgf - b wild - type and pdgf - m2 mutant by high resolution lc / ms demonstrated that for wild - type pdgf , different isoforms containing up to 6 carbohydrate residues were detected , wherein the content of the isoform containing three carbohydrate residues was the highest . meanwhile , glycosylation could hardly be detected for the pdgf - m2 ( m2 ) mutant ( as shown in fig9 ). although the specific embodiments of the invention have been described in detail , those skilled in the art will appreciate that in accordance with all the teachings which have been disclosed , various modifications and substitutions may be made to those details and these changes are all within the scope of the present invention . the scope of the invention is given by the appended claims and any equivalents thereof . 1 . ross , r ., glomset , j ., kariya , b ., and harker , l . 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