PMID- 2687112
OWN - NLM
STAT- MEDLINE
DCOM- 19900116
LR  - 20190707
IS  - 0378-1119 (Print)
IS  - 0378-1119 (Linking)
VI  - 83
IP  - 1
DP  - 1989 Nov 15
TI  - Overexpression of native human beta 2-microglobulin in Escherichia coli and its 
      purification.
PG  - 117-24
AB  - beta 2-Microglobulin (beta 2M), the small subunit of human leukocyte antigen 
      (HLA) class-I proteins, has been synthesized in Escherichia coli and purified in 
      mg amounts. A beta 2m cDNA clone was fused in-frame behind DNA encoding the 
      signal sequence for the outer membrane protein, OmpA. Three different 
      constructions were made, whose products differed by the insertion of either an 
      extra Ala residue, the hexapeptide AEFLEA [single-letter amino acid (aa) code], 
      or no aa between the OmpA signal sequence and beta 2M-coding sequence. All three 
      protein products were correctly processed by bacterial signal peptidase, as 
      determined by N-terminal sequencing, and all three were secreted as soluble 
      proteins into the periplasmic space. However, the signal sequence of the 
      preprotein with the inserted hexapeptide, AEFLEA, was cleaved to a much greater 
      degree than the other two preproteins. When there was no insertion, the mature 
      protein was identical to human beta 2M, as analyzed by sodium dodecyl 
      sulfate-polyacrylamide-gel electrophoresis, circular dichroism, and native 
      isoelectric focusing. This 'bacterial beta 2M', radiolabeled with Bolton-Hunter 
      reagent, was able to exchange into papain-solubilized HLA-B7, as determined by 
      Sephadex G-75 chromatography and immune precipitation, indicating that bacterial 
      beta 2M could complex with the heavy chain of HLA-B7.
FAU - Parker, K C
AU  - Parker KC
AD  - Department of Biochemistry and Molecular Biology, Howard Hughes Medical 
      Institute, Harvard University, Cambridge, MA 02138.
FAU - Wiley, D C
AU  - Wiley DC
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - Netherlands
TA  - Gene
JT  - Gene
JID - 7706761
RN  - 0 (Membrane Proteins)
RN  - 0 (Recombinant Proteins)
RN  - 0 (beta 2-Microglobulin)
RN  - EC 3.4.- (Endopeptidases)
RN  - EC 3.4.21.- (Serine Endopeptidases)
RN  - EC 3.4.21.89 (type I signal peptidase)
SB  - IM
MH  - Blotting, Western
MH  - Chromatography, Gel
MH  - Endopeptidases/physiology
MH  - Escherichia coli/*genetics
MH  - Gene Expression/*physiology
MH  - Humans
MH  - *Membrane Proteins
MH  - Plasmids
MH  - Recombinant Proteins/biosynthesis
MH  - *Serine Endopeptidases
MH  - beta 2-Microglobulin/biosynthesis/*genetics
EDAT- 1989/11/15 00:00
MHDA- 1989/11/15 00:01
CRDT- 1989/11/15 00:00
PHST- 1989/11/15 00:00 [pubmed]
PHST- 1989/11/15 00:01 [medline]
PHST- 1989/11/15 00:00 [entrez]
AID - 0378-1119(89)90409-5 [pii]
AID - 10.1016/0378-1119(89)90409-5 [doi]
PST - ppublish
SO  - Gene. 1989 Nov 15;83(1):117-24. doi: 10.1016/0378-1119(89)90409-5.