臺灣博碩士論文加值系統

以重組DNA技術在大腸桿菌中製備人類白血癌抑制因子人類白
血癌抑制因子(human leukemia inhibitory factor;hLIF)是一種具有多
功能的生長因子,可在不同的細胞中,具有特定的功能. pET29b(+)表現載
體(expression vector)包括S.Tag和His.Tag特殊的核甘酸序列,因此所表
現出來的蛋白質具有S.Tag和His.Tag特殊的胺基酸序列,有別於其他載體
系統,用以偵測和純化由表現載體製造的蛋白質. 基於此緣故,在實驗室中
我們利用大腸桿菌(E.coli)大量製備rhLIF,由實驗結果發現以BLR(DE3)
pLysS菌株表現量最好,並且在加入IPTG(isopropyl beta-D-
thiogalactopyranoside)誘發3小時候rhLIF的產率最大,經過3小時候表現
量不再明顯地增加.純化的過程中首先利用His.Tag鎳離子箝合層析管柱純
化rhLIF,以蛋白質電泳膠片染色結果發現尚存留其他蛋白質,純化效果不
理想.隨後我們嘗試先以S-蛋白質親和性管柱純化rhLIF,以蛋白質電泳膠
片染色結果顯示仍含有其他蛋白質.經過透析後以人類凝血酵素分解脢(
humanthrombin)去切除S-蛋白質部分,最後再利用His.Tag鎳離子箝合層析
管柱純化rhLIF並且以蛋白質電泳膠片銀染色可見高純度的rhLIF.結合S-
蛋白質親和性管柱層析法和His.Tag鎳離子箝合層析管柱層析法可以快速
得到高純度的rhLIF.以微量BCA定量法測得rhLIF的產量約為六公升BLR(
DE3)pLysS菌株可產生84.288 ug.進一步以TF-1細胞進行生物活性分析(
biological activity assay;MTT assay),結果發現純化的rhLIF並不具生
物活性,可能的原因是製備出來的rhLIF胺基酸序列錯誤,末端具有額外的
六個組胺酸殘基 (histidine residues)影響蛋白質三級構造和功能,在純
化的過程中雙硫鍵被破壞失去生物活性或TF-1細胞不適合用於rhLIF的生
物活性分析等等.

Expression of Recombinant Human Leukemia Inhibitory Factor in
Escherichia coliHuman leukemia inhibitory factor is a multi
functional growth factor. The pET29b(+)expression vector
includes S.Tag and His.Tag nucleotide sequence, encoding a
15-amino acid S-peptide and a histidine hexmar. Protein tagged
with these two polypeptidesare easy to be detected and purified.
For this reason, we utilize Escherichia coli toproduce
recombinant human leukemia inhibitory factor in labatory. The
results indicatethat rhLIF is expressed in a large amount in
strain BLR(DE3)pLysS rather than in the other two tested
strains. The production of rhLIF reaches to maximum at 3 hours
after IPTG induction. The strategy to prepare rhLIF is briefly
described below. The crudebacterica lysated was applied onto a
His.Tag nickel-ion chelating column and the boundproteins were
eluted with buffer containing 1M imidazole. The elute, however,
is heterogenous. The S.Tag affinity column chromatography was
therafter employed. The purity of target protein was
chamatically improved albeit minor heterogeneity stillpersist.
The proteins eluted from S.Tag column were then cleaved by
thrombin and thenpass through a His.Tag column. Finally, the
target protein can be purified to its homogenity. The yield is
about 84.288 ug from 6 liters of BLR(DE3)pLysS culture.
Thebiological activity of the purified protein was analysed as
its proliferating activityon TF-1 cells. The LIF-like activity.
However, was not detected. The possible reasons leading to lose
activity of the purified proteins will be discussed in later
part of this thesis.