The plasmid containing human insulin-like growth factor-II(IGF-II) cDNA was modified to delete nontranslated region and portions of coding region for IGF-II at both 5'-and 3'-end of the cDNA sequence. A gene encoding mature IGF-II was prepared from the modified cDNA sequence and double-stranded synthetic linkers. The synthetic linkers was designed to resupply the missing coding information for mature IGF-II polypeptede. For the expression of mature IGF-II in $\underline{E}$. $\underline{coli}$, the expression vector, pSIG was constructed by placing the gene encoding mature IGF-II, in correct frame, just after translational initiation codon, ATG, of pASI. In the pSIG, the IGF-II gene was located under the control of lamda phage $P_L$ promoter. The $\underline{E}$. $\underline{coli}$ strain M5248 harbouring pSIG was induced to express human IGF-II polypeptide by rapid shifting of temperature (32˚-->42℃). However, recombinant IGF-II was not detectable by Commassie Blue staining of the total cellular lysates which were subjected to SDS-polyacrylamide gel electrophoresis.
In order to improve expression of IGF-II gene in $\underline{E}$. $\underline{coli}$, we decided to fuse the gene to 3'-end of $\underline{lac}$ Z gene. We constructed another expression plasmid, pYRM-$II_4$ by inserting the coding sequence for Asp-Pro-Met-IGF-II into ClaI site within $\underline{lac}$z gene of pCTIO. Thus, the fused lacZ'-IGF-II gene was located under the control of $\underline{tac}$ promoter of pCTIO. The mature IGF-II have been overproduced as C-terminal part of β-galactosidase composed of N-terminal 280 amino acids. The $\underline{E}$. $\underline{coli}$ JM109 cells harbouring pYRM-$II_4$ was induced to express the fused gene by 2 mM IPTG at early log phase. The level of expression of IGF-II was observed 6 hours after induction. It was estimated by densitometric scanning that the recombinant fusion protein accounted about 25% of the total cellular proteins. The IGF-II fusion protein formed inclusion bodies, more and less homogenous protein aggregates, inside the cells as shown by transmission electron micrograph.
The inclusion bodies containing IGF-II were pelleted readily by low speed centrifugation (1000 × g for 10 min), leaving other soluble protein of the $\underline{E}$. $\underline{coli}$ cellular lysates in supernatant. Contaminant proteins co-purified with the inclusions were partially removed by washing with 4 M urea. The 4 M urea-washed inclusion bodies were solubilized in 0.1M Tris-HCl, pH 8.0 containing 8 M urea and 35 mM 2-mercaptoethanol. The solubilized crude the IGF-II fusion protein were fractionated on Sepharose 6B gel filtration chromatography, and purified to Homogeniety using preparative polyacryamide gel electrophoresis.
In order to isolated IGF-II from its fusion protein, the unique sequence Asp-Pro which was located between truncated β-gal and IGF-II, was cleaved off by acid treatment. However, several nonspecific peptide bond cleavages other than the unique Asp-Pro bond were observed. In addition to these nonspecific cleavages, the cleavage was not efficient.
Next, we tried to releave IGF-II by treatment of the IGF-II fusion protein with CNBr. The Met residue was designed to place at the begining of the mature IGF-II. All the Met-X bonds except Met-Cys and Met-Ser in β-gal was cleaved off efficiently, and putative mature IGF-II was observed by 18% SDS-PAGE analysis of the cleaved products. The apparent molecular weight of the IGF-II identified to be about 6 KDa on the 18% SDS-PAGE. The putative IGF-II was confirmed to be mature IGF-II by its ability to bind to anti-IGF-I antibody.
Attempts to purify mature IGF-II out of the cleaved IGF-II fusion protein was done using preparative reverse-phase HPLC. However, aggregation of the proteins eluted from the column was occurred, which was shown by urea-SDS-PAGE analysis. The purified IGF-II fusion protein was used as a immunogen, and the immunized spleen cell was hybridized with mouse Sp2/0-Ag14 myeloma cell. The production of monoclonal antibodies against the fusion protein was screened by using ELISA. The selected monclonal antibodies were identified as IgGl by Ouchterlony double immuno diffusion method.
본 논문에서는 인간 insulin-like growth factor II(IGF-II)를 대장균에서 대량생산하고, 재조합 IGF-II 융합단백질을 분리정제 하고 이의 생화학적 특성에 관하여 연구하였다. 인간 IGF-II cDNA와 합성된 oligodeoxynucleotide linker들을 이용하여 성숙된 IGF-II를 전사시키는 유전자를 만들고, 이 유전자를 pASI 발현벡타의 ATG 바로 뒤에 연결시켜 lamda phage P promoter에 조절하에 성숙한 IGF-II를 대장균에서 IGF-II를 발현하도록하였다. 이렇게 재조합된 발현벡터를 갖고 있는 대장균을 42˚에서 배양하여 IGF-II 유전자를 발현시켰을 때, 재조합 IGF-II가 거의 생산되지 않았음을 전기영동의 분석으로 확인 할 수 있었다. 대장균에서의 IGF-II의 효율적인 발현을 위한 방편의 하나로서, IGF-II 유전자를 lacZ 유전자와 융합시켜, 이 융합된 유전자가 안정된 단백질 형태인 β-galactosidase와 융합된 IGF-II를 전사하도록 하였다. IGF-II 유전자를 β-galactosidase (β-gal)의 카복실잔기 쪽의 280 아미노산을 전사하는 잘려진 lacZ 유전자와 융합시키고, 이를 tac promoter의 조절하에 대장균에서 발현시켜 보면, IGF-II 융합단백질이 대량생산되며 이 융합단백질은 분자량이 약 47000 임을 전기영동결과로 확인할 수 있었다. 또한, IGF-II 융합단백질이 전체 세포 단백질의 약 25% 를 차지하고 있음을 염색한 젤의 densitometric scanning 의로 확인하였다. 이 IGF-II 융합단백질이 세포내에서 inclusion bodies를 형성하고 있음을 전자 현미경으로 관찰하였으며, 이 단백질을 포함하고 있는 inclusion bodies 는 저속의 원심분리 (1000 × g에서 10 분)로 쉽게 세포질내 가용성 단백질들과 분리되어, 침전물로 얻을 수 있었다. 이 침전물에 오염된 다른 단백질들은 4 M urea로 씻어 부분적으로 제거하였고, IGF-II 융합단백질은 8 M urea와 35 mM 2-mercaptoethanol이 포함된 0.1 M TrisHCl, pH 8.0 에서 가용화 시킬 수 있었다. 가용화된 단백질 용액을 Sepharose 6B gel 크로마토그라피와 분리정제용 전기영동 방법으로 IGF-II 융합단백질을 순수하게 분리하였다. 분리된 융합단백질로 부터 IGF-II를 얻어 내기 위하여, β-gal""과 IGF-II 사이에 위치한 유일한 Asp-Pro peptide 결합을 여러가지 산으로 처리함으로써 특이적으로 가수분해시키고자 하였다. 그러나, Asp-Pro peptide 결합이외의 다른 여러 peptide 결합들도 비특이적으로 잘려진 것과 특이적 가수분해 반응의 효율이 저조함을 전기영동 결과로서 알 수 있었다. 따라서, IGF-II 융합단백질의 IGF-II 아미노산 잔기의 바로 앞에 위치한 Met을 CNBr로 잘라 냄으로써 IGF-II를 얻도록 하였다. CNBr에 의하여 잘려 나온 IGF-II는 SDS 전기영동 방법으로 분자량이 약 6000 정도 임을 알 수 있었으며, anti-IGF I antibody에 의하여 인식되어 짐을 Western blotting 방법으로 확인할 수 있었다. 분리된 IGF-II 융합단백질을 항원으로 사용하여 이에 대한 단일항체를 제조하였으며 이 단일항체는 Ouchterlony double immunodiffusion 방법에 의하여 Ig Gl 임을 확인할 수 있었다.
