간염 B 형 바이러스 표면항원 대장균 내 발현

최영철,김지영,유명희,한문희 ( Young Chul Choi,Ji Young Kim,Myeong Hee Yu,Moon H . Han ) 생화학분자생물학회 1986 BMB Reports Vol.19 No.4

Three different HBsAg sequences encoding for S region, pre-S2 plus S region, and pre-S2 fused with a portion of β-galactosidase have been expressed in E. coli under the control of lac promoter. When the S region containing HBsAg genes were expressed constitutively in E. coli HB101, the growth inhibition of recombinant cells was observed. However, the constitutive expression of pre-S2 fused with apeptide (N-terminal portion of β-galactosidase) was not detrimental to the E. coli HB101 cells. The antigenicity of the hybrid pre-S2 protein seems to be conformation-independent: treatment of 2-mercaptoethanol or 2-mercaptoethanol/SDS did not abolish the antigenicity.

Expression of Hepatitis B Virus Surface Antigen Gene in E. coli

최영철,김지영,유명희,한문희,Choi, Young-Chul,Kim, Ji-Young,Yu, Myeong-Hee,Han, Moon-H. 생화학분자생물학회 1986 한국생화학회지 Vol.19 No.4

간염 B형 바이러스의 표면항원 유전자중에서 S부위, pre-S2와 S부위 그리고 $\beta$-galactosidase의 일부와 연결된 pre-S2 부위를 포함하는 세가지의 유전자 서열들을 lac promoter의 조절하에 대장균 내에서 발현시켰다. S부위를 포함하는 HBsAg 유전자가 E. coli HB101균주 내에서 지속적으로 발현될 경우에는 재조합 균주의 성장이 심하게 억제되었다. 그러나, $\alpha$-peptide ($\beta$-galactosidase의 N-말단부위)와 연결된 pre-S2 부위의 지속적인 발현은 E. coli HB101균주의 성장에 해로운 영향을 미치지 않았다. 또한, pre-S2 융합단백질의 항원성은 conformation과 무관한 것으로 나타났다. 2-mercaptoethanol 또는 2-mercaptoethanol/SDS의 처리후에도 항원성이 보존되었다. Three different HBsAg sequences encoding for S region, pre-S2 plus S region, and pre-S2 fused with a portion of $\beta$-galactosidase have been expressed in E. coli under the control of lac promoter. When the S region containing HBsAg genes were expressed constitutively in E. coli HB101, the growth inhibition of recombinant cells was observed. However, the constitutive expression of pre-S2 fused with $\alpha$-peptide (N-terminal portion of $\beta$-galactosidase) was not detrimental to the E. coli HB101 cells. The antigenicity of the hybrid pre-S2 protein seems to be conformation-independent: treatment of 2-mercaptoethanol or 2-mercaptoethanol/SDS did not abolish the antigenicity.

Expression of a DNA Sequence Encoding for the Pre-S2 Region of Hepatitis B Virus in E. coli

최영철,김지영,유명희,한문희,Choi, Young-Chul,Kim, Ji-Young,Yu, Myeong-Hee,Han, Moon-H. 생화학분자생물학회 1986 한국생화학회지 Vol.19 No.4

간염 B형 바이러스의 pre-S2 부위를 포함하는 DNA 서열을 pORF1 플라스미드의 ompF와 lacZ 유전자 사이에 끼워 넣어 pHBSC20 재조합 플라스미드를 제조하였다. 플라스미드 pHBSC20를 갖는 E. coli TK1046 균주내에서 OmpF와 $\beta$-galactosidase사이에 pre-S2 부위를 갖는 "tribrid" 단백질이 생성되었다. 이 융합단백질은 SDS-polyacrylamide gel 전기영동에 의해 확인되었고 pre-S2 부위의 항원성은 immunoblot 분석방법에 의해 확인되었다. 융합단백질의 양은 Coomassie blue로 염색한 gel을 densitometer로 측정한 결과 전체 박테리아 단백질중 1-5%를 차지하는 것으로 나타났다. 따라서 pHBSC20 플라스미드를 갖는 박테리아는 pre-S2-$\beta$-galactosidase 융합 단백질을 다량 만들어 낼 수 있으며 이 중에서 pre-S2 부위는 HBV 백신을 생산하는데 유용할 것으로 보인다. A DNA sequence involving pre-S2 region of Hepatitis B virus (HBV) was inserted between ompF and lacZ gene of the plasmid pORF1 to give a recombinant plasmid pHBSC20. In the plasmid pHBSC20 harboring E. coli TK1046 cells, a "tribrid" protein with the translation product of the insert sandwiched between OmpF and $\beta$-galactosidase was produced. The fusion protein was detected by SDS-polyacrylamide gel electrophoresis and the antigenicity of the pre-S2 region was determined by immunoblot analysis. The amount of the fusion protein was estimated as much as 1-5% of the total bacterial protein by densitometric scanning of the Coomassie blue-stained gel. Thus, bacteria transformed with pHBSC20 can provide abundant source of pre-S2-$\beta$-galactosidase fusion protein in which the pre-S2 region may be useful for the production of HBV vaccine.

감염 B 형 바이러스의 pre - S2 부위를 code 하는 DNA 서열의 대장균내 발현

최영철,김지영,유명희,한문희 ( Young Chul Choi,Ji Young Kim,Myeong Hee Yu,Moon H . Han ) 생화학분자생물학회 1986 BMB Reports Vol.19 No.4

A DNA sequence involving pre-S2 region of Hepatitis B virus (HBV) was inserted between ompF and lacZ gene of the plasmid pORF1 to give a recombinant plasmid pHBSC20. In the plasmid pHBSC20 harboring E. coli TK1046 cells, a $quot;tribrid$quot; protein with the translation product of the insert sandwiched between OmpF and β-galactosidase was produced. The fusion protein was detected by SDS-polyacrylamide gel electrophoresis and the antigenicity of the pre-S2 region was determined by immunoblot analysis. The amount of the fusion protein was estimated as much as 1-596 of the total bacterial protein by densitometric scanning of the Coomassie blue-stained gel. Thus, bacteria transformed with pHBSC20 can provide abundant source of pre-S2-β-galactosidase fusion protein in which the pre-S2 region may be useful for the production of HBV vaccine.

대장균에서 융합단백질의 대량생산을 위한 베타갈락토시다제 폴리펩티드 길이의 최적화

최영철,이상철,한문희,유명희 ( Young Chul Choi,Sang Chul Lee,Moon H . Han,Myeong Hee Y ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.1

Plasmid vectors for cloning and directing the synthesis of large amounts of fusion proteins in E. coli were constructed. These plasmids carried tac promoter, the coding sequence of amino terminal portion of β-galactosidase, and the polylinker region at the 3`-end of truncated lacZ. When E. coli MC1061 cells were transformed with these plasmids, 25-60% of the total cellular proteins were represented by the lacZ polypeptides. The overproduced proteins formed inclusion bodies inside the cells. When the amounts of recombinant proteins produced by these plasmids were examined, the higher yield was observed with those of shorter lacZ polypeptides. The length of recombinant polypeptide with the maximum yield (60%) was about 280 amino acids long. However, when the length of β-galactosidase decreased to 200 residues as in pCT30 vector, the yield of recombinant proteins decreased dramatically. When the length of polypeptide increased again to 260 residues by fusing pre-S2 sequence of hepatitis B virus to lacZ sequence in pCT30, the yield of fusion polypeptides increased to over 50% of total cellular proteins.

Oligonucleotide-directed Mutagenesis of Two Methionine Residues in Recombinant ${\beta}$-galactosidase-preS2 Fusion Proteins

박종광,이상철,최영철,한문희,유명희,Park, Chong-Kwang,Lee, Sang-Chul,Choi, Young-Chul,Han, Moon-H.,Yu, Myeong-Hee 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.1

플라스미드 pCTHB20에 의해 형질전환된 대장균 JM109 균주는 베타갈락토시다제-프리 S2 융합 단백질을 총 세포 단백질의 50% 정도로 생산하였다 (Choi et al., 1988). 이 융합 단백질로부터 시안브롬을 사용하여 프리 S2 펩티드를 절단하여 정제하는 것을 보다 더 용이하게 하기 위해 베타갈락토시다제 유전자 (lacZ) 서열을 재배열하여 pCTHB30을 제조하였으며, 또한 lacZ 서열 내부에 존재하는 메치오닌 코돈을 올리고뉴클레오티드에 의한 변이유도 방법을 사용하여 발린코돈으로 치환하였다. 플라스미드 pCTHB2에 존재하는 lacZ 서열을 포함하는 EcoRI-XbaI DNA 절편을 분리하여 파지 M13 mp18 백터의 멀티크로닝 부위에 삽입하였고, 원하는 변이를 포함하는 두개의 상보쇄 서열 (각각 17-mer)을 합성하여 동시에 프라이머로 사용하였다. 변이클론들은 $^{32}P$로 표지된 변이 올리고뉴클레오티드를 프로브로 사용하여 DNA 하이브라디제이션 방법에 의해 선별하였으며, 치환된 뉴클레오티드는 DNA 염기서열 분석에 의해 확인하였다. 변이 lacZ 서열로 치환하여 제조된 pCMHB20 및 PCMHB3에 의해 형질전환된 대장균 JM109 균주들도 pCTHB20 및 PCTHB30을 포함하는 균주와 거의 같은 효율로 프리 S2 서열을 지닌 융합 단백질을 생산하였다. E. coli JM109 cells harboring pCTHB20 plasmid overproduced ${\beta}$-galactosidase-preS2 ($\beta$ gal-preS2) fusion proteins with a yield of 50% of total cellular proteins (Choi et al., 1988). In order to facilitate the isolation of preS2 peptide out of the fusion protein after CNBr cleavage, the lacZ sequence was rearranged to construct pCTHB30 plasmid. Two internal methionine codons in the lacZ region of pCTHB20 and pCTHB30 was also substituted with valine codons by oligonucleotide-directed mutagenesis. The EcoRI-XbaI restriction fragment of lacZ was isolated from the plasmid pCTHB20 and was inserted into multicloning site of M13 mp18 vector. Two synthetic complementary oligonucleotides (17-mer each) with desired nucleotide changes were used simultaneously as primers in synthesizing a complementary strand. Mutant clones were screened by DNA hybridization with probes of $^{32}P$-labeled mutagenic oligonucleotides, and the nucleotide substitutions were confirmed by DNA sequencing. E. coli JM109 cells transformed with pCMHB20 and pCMHB30 in which the lacZ regions were substituted with the mutant counterpart produced ${\beta}$ gal-preS2 fusion proteins as efficiently as those with pCTHB20 and pCTHB30.

재조합 베타갈락토시다제 - 프리 S2 융합단백질에 존재하는 두 개의 메치오닌 위치에 올리고뉴클레오티드를 이용한 변이유도

박종광,이상철,최영철,한문희,유명희 ( Chong Kwnag Park,Sang Chul Lee,Young Chul Choi,Moon H . Han,Myeong Hee Yu ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.1

E. coli JM109 cells harboring pCTHB20 plasmid overproduced β-galactosidase-preS2 (βgal-preS2) fusion proteins with a yield of 50% of total cellular proteins (Choi et al., 1988). In order to facilitate the isolation of preS2 peptide out of the fusion protein after CNBr cleavage, the lacZ sequence was rearranged to construct pCTHB30 plasmid. Two internal methionine codons in the lacZ region of pCTHB20 and pCTHB30 was also substituted with valine codons by oligonucleotide-directed mutagenesis. The EcoRI-XbaI restriction fragment of lacZ was isolated from the plasmid pCTHB20 and was inserted into multicloning site of M13 mp18 vector. Two synthetic complementary oligonucleotides (17-mer each) with desired nucleotide changes were used simultaneously as primers in synthesizing a complementary strand. Mutant clones were screened by DNA hybridization with probes of ^(32)P-labeled mutagenic oligonucleotides, and the nucleotide substitutions were confirmed by DNA sequencing. E. coli JM109 cells transformed with pCMHB20 and pCMHB30 in which the lacZ regions were substituted with the mutant counterpart produced βgal-preS2 fusion proteins as efficiently as those with pCTHB20 and pCTHB30.

Optimization of the ${\beta}$-galactosidase Polypeptide Length for the Overproduction of Fusion Proteins in E. coli.

최영철,이상철,한문희,유명희,Choi, Young-Chul,Lee, Sang-Chul,Han, Moon-H.,Yu, Myeong-Hee 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.1

대장균 내에서 융합단백질을 다량 생산하기 위한 발현벡터들이 개발되었다. 이들 플라스미드 벡터들은 tac 전사활성화 서열, 베타갈락토시다제의 아미노 말단을 코우드하는 뉴클레오티드 서열 그리고 그의 3'말단에 클로닝 부위로서 폴리링커를 지니고 있다. 대장균 MC1061 균주를 이들 플라스마드로 형질전환 시키고 재조합 단백질의 생산을 유도하였을 때 절단된 베타갈락토시다제 폴리펩티드의 양은 전체 세포단백질의 25-60% 정도가 되었다. 이렇게 생산된 재조합 단백질들은 세포내에서 함유체 (inclusion body) 상태로 축적되었다. 이들 플라스미드에 의해 생산되는 폴리펩티드의 수율을 조사하였을 때 길이가 짧을수록 수율이 올라감을 알 수 있었는데, 길이가 280개 아미노산 정도일 때 최대의 수율(60%)을 나타냈다. 하지만 pCT30 플라스미드에서와 같이 길이가 200개 아미노산 정도로 너무 짧아지면, 수율이 현저하게 감소하였다. 이 pCT30 플라스미드의 베타갈락토시다제 서열의 3'말단에 간염 바이러스의 pre-S2 서열을 연결시켜, 생산되는 융합 폴리펩티드 길이를 260개 아미노산으로 늘어나게 하였을 때 융합 단백질의 수율은 다시 총 세포 단백질의 50% 이상으로 증가하였다. Plasmid vectors for cloning and directing the synthesis of large amounts of fusion proteins in E. coli were constructed. These plasmids carried tac promoter, the coding sequence of amino terminal portion of ${\beta}$-galactosidase, and the polylinker region at the 3'-end of truncated lacZ. When E. coli MC1061 cells were transformed with these plasmids, 25-60% of the total cellular proteins were represented by the lacZ polypeptides. The overproduced proteins formed inclusion bodies inside the cells. When the amounts of recombinant proteins produced by these plasmids were examined, the higher yield was observed with those of shorter lacZ polypeptides. The length of recombinant polypeptide with the maximum yield (60%) was about 280 amino acids long. However, when the length of ${\beta}$-galactosidase decreased to 200 residues as in pCT30 vector, the yield of recombinant proteins decreased dramatically. When the length of polypeptide increased again to 260 residues by fusing pre-S2 sequence of hepatitis B virus to lacZ sequence in pCT30, the yield of fusion polypeptides increased to over 50% of total cellular proteins.